Treating respiratory conditions

ABSTRACT

A method for treating a respiratory conditions comprises applying peripheral blood from a patient or subject to an apheresis column loaded with a solid support comprising one or more binding reagents capable of specifically binding to a chemokine receptor, optionally the chemokine receptor CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 immobilized directly or indirectly on the support thus removing one or more chemokine receptor, optionally CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells from the peripheral blood of the patient or subject. Various companion diagnostic methods and useful binding reagents are also described.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. patent application Ser. No.14/105,628, filed on Dec. 13, 2013, now issued as U.S. Pat. No.9,726,666, which is a continuation-in-part of International PatentApplication No. PCT/GB2012/051357, filed on Jun. 13, 2012, which claimspriority to U.S. Provisional Patent Application No. 61/496,442, filed onJun. 13, 2011. U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051349, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,167, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051348, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,288, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051351, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,242, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051350, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,209, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051355, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,195, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051345, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,228, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051352, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,264, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051346, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,184, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051353, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,329, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051356, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,377, filed on Jun. 13, 2011.U.S. patent application Ser. No. 14/105,628 is also acontinuation-in-part of International Patent Application No.PCT/GB2012/051354, filed on Jun. 13, 2012, which claims priority to U.S.Provisional Patent Application No. 61/496,352, filed on Jun. 13, 2011.The entire contents of each of these applications are fully incorporatedherein by reference.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 21, 2017, isnamed P81602729US01-211226-9003-US01-SEQ-LIST-06-21-17.txt, and is26,466 bytes in size.

FIELD OF THE INVENTION

The various embodiments of the present invention relate to products forand methods of treating inflammatory conditions, such as respiratoryconditions, in particular sarcoidosis and Chronic Obstructive PulmonaryDisease (COPD). Companion diagnostics are also described.

BACKGROUND OF THE INVENTION

Respiratory tract disorders such as asthma and chronic obstructivepulmonary disease (COPD) are typically characterised by breathingdifficulties as a result of reduced airflow to the lungs. These symptomsare often caused by inflammation of the airways; for example, chronicbronchitis is a form of COPD associated with excessive inflammation ofthe bronchi.

In addition to breathing difficulties, airway obstruction and associatedinflammation can cause progressive lung damage. In the case of patientswith COPD, permanent narrowing of the airways can lead to complicationssuch as chest infections, heart failure and ultimately pulmonaryfailure.

COPD is a very common respiratory disease and in the majority of cases,smoking is the cause.

Patients are typically treated using inhalers containing“bronchodilator” drugs. However, these are of limited use for patientswith permanently-constricted airways and/or end-stage diseasecharacterised by extensive damage to the lungs.

Apheresis is a treatment used for depletion of blood components, such asantibodies, low-density lipoproteins (LDL) and blood cells.Leukapheresis is the apheresis treatment used for removal of white bloodcells, leukocytes. The patient is connected to an extracorporeal bloodcirculating system; the blood is drawn from a vein in one arm, passedthrough a column device and returned into the other arm of the patient.WO2010/029317 describes apheresis columns useful for treatinginflammatory conditions including a chemokine immobilised on a solidsupport.

SUMMARY OF THE INVENTION

Chemokines are a class of cytokine molecules involved in cellrecruitment and activation in inflammation. Chemokines cause chemotaxisand activation of various subpopulations of cells in the immune system.The activity of chemokines is mediated primarily through tight bindingto their receptors on the surface of leukocytes. In certain embodimentsthe present invention is based on the realisation that the interactionbetween chemokines and cells expressing their receptors may be exploitedfor the treatment of respiratory conditions, in particular sarcoidosisand Chronic Obstructive Pulmonary Disease (COPD). In particular, variousrespiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD) include an inflammatory component.Whilst sarcoidosis is predominantly considered a respiratory condition,general treatment of sarcoidosis affecting any organ of the body may bepossible within the scope of the various embodiments of the presentinvention. The inventors have determined that targeting increasedrecruitment of specific chemokine receptor-expressing cells to the siteof inflammation presents a new therapeutic approach to treat suchconditions. Moreover, in such conditions, chemokine receptor expressionon each cell may be increased again providing a therapeutic approach totreat such conditions. It is shown herein that subjects suffering fromrespiratory conditions such as sarcoidosis exhibit increased frequencyof chemokine receptor expressing cells in the peripheral blood. Subjectswith sarcoidosis exhibit increased frequency of CCR1 expressing cellssuch as CCR1 expressing monocytes, compared to healthy controls. It isalso shown herein that the CCR1 expressing cells can be removed using asuitable binding reagent, in particular RANTES (in biotinylated form)immobilized on a suitable matrix. Similarly, it is shown herein that themonocytes also express CCR2. The CCR2 expressing monocytes can bedepleted in sarcoidosis patients using a suitable binding reagent, inparticular MCP-1, in biotinylated form, immobilized on a suitablematrix. It is also shown herein that subjects suffering from respiratoryconditions such as sarcoidosis exhibit increased frequency of CCR7expressing cells such as CCR7 expressing lymphocytes, and also centralmemory T cells, compared to healthy controls. It is also shown hereinthat the CCR7 expressing cells can be removed using a suitable bindingreagent, in particular MIP3b (in biotinylated form) immobilized on asuitable matrix.

Thus, in certain embodiments the invention serves to reduce therecruitment of inflammatory leukocytes, which express characteristicchemokine receptors, and possibly express characteristic chemokinereceptors at increased levels, to sites of inflammation linked torespiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD). This is achieved using specificbinding reagents to capture specific chemokine receptor-expressinginflammatory leukocytes from the patient. Accordingly, in certainembodiments the invention provides in a first aspect a method fortreating respiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD) comprising applying peripheralblood from a patient to an apheresis column loaded with a solid supportcomprising one or more binding reagents capable of specifically bindingto one or more chemokine receptors, in particular the chemokinereceptors CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7, immobilizeddirectly or indirectly on the support thus removing one or morechemokine receptor, in particular one or more of CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7, expressing cells from the peripheral blood ofthe patient. The peripheral blood from which the chemokine receptorexpressing cells have been removed may then be returned to the patientin order to complete the treatment. The invention may thus rely on acontinuous extracorporeal circuit in some embodiments. Alternatively, incertain embodiments the invention may comprise steps of obtainingperipheral blood from the patient, applying the peripheral blood to thecolumn and subsequently returning the peripheral blood from which thechemokine receptor expressing cells have been removed to the patient.

As shown herein, suitable binding reagents can be immobilized onto asolid support, either directly or indirectly, to generate an apheresiscolumn suitable for capturing relevant chemokine receptor-expressingcells. Where increased levels of chemokine receptor expression areobserved, such cells may be preferably removed from the peripheral bloodusing the columns of the various embodiments of the invention. Thus, themethods of the various embodiments of the invention may preferablytarget one or more of CCR2hi, CCR1hi, CCR3hi, CCR5hi, CXCR1hi, CXCR2hiand/or CCR7hi cells as defined herein for removal from the peripheralblood. “High” expression may be determined according to standard flowcytometry techniques. The level is measured relative to levels ofexpression of the chemokine receptor in cells taken from a healthysubject. The attached FIG. 17 provides an example of a gating strategy.

Herein, reference to CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 isintended to encompass selection of any one or more, up to all, of thechemokine receptors listed. In addition, the combination of CCR2, CCR1,CCR3, CCR5, CXCR1 and/or CXCR2 is explicitly contemplated as a separategrouping, to include any one or more of CCR2, CCR1, CCR3, CCR5, CXCR1and CXCR2.

In other embodiments the invention further provides a binding reagentcapable of specifically binding to one or more chemokine receptors, inparticular to a chemokine receptor/the chemokine receptor CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7, for use in the treatment ofrespiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD), wherein the one or more bindingreagents is immobilized, directly or indirectly, on a solid supportcontained within an apheresis column, to which is applied peripheralblood from a patient thus removing one or more chemokine receptor/CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells from theperipheral blood of the patient. In certain embodiments the inventionalso provides for use of one or more binding reagents capable ofspecifically binding to a chemokine receptor/the chemokine receptorCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 for use in themanufacture of an apheresis column for treatment of respiratoryconditions, in particular sarcoidosis and Chronic Obstructive PulmonaryDisease (COPD), wherein the one or more binding reagents is immobilizedon a solid support contained within the apheresis column, to which isapplied peripheral blood from a patient thus removing one or more ofchemokine receptor/CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expressing cells from the peripheral blood of the patient.

All embodiments described in respect of the methods of treatment of thevarious embodiments of the invention apply to these aspects mutatismutandis and are not repeated for reasons of conciseness. Thus, thefollowing discussion made with reference to the various embodiments ofthe methods of treatment is also applicable to the medical use aspectsof the invention.

In certain embodiments the invention aims to treat a range ofrespiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD). By treatment is meant a reductionin the specific chemokine receptor expressing cells in the peripheralblood of the patient. The reduction may comprise a reduction in cellsthat express chemokine receptors, in particular one or more of CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7, at increased levels indiseased patients. The patient is typically a human patient but the termpatient may include both human and non-human animal subjects in someembodiments. In the context of the various embodiments of the presentinvention, this typically involves a reduction in one or more of CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, such as oneor more of CCR2^(hi), CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi),CXCR2^(hi) and/or CCR7^(hi) expressing cells, in the peripheral blood ofthe patient. The CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expressing cells comprise, consist essentially of or consist ofmonocytes, macrophages, lymphocytes, in particular T-lymphocytes, and/oreosinophils in certain embodiments. In specific embodiments, the cellsremoved in order to treat respiratory conditions such as sarcoidosiscomprise monocytes, in particular CCR1 and/or CCR2 expressing monocytes.In other embodiments cells removed in order to treat respiratoryconditions such as sarcoidosis comprise lymphocytes, specifically Tlymphocytes such as central memory T cells, in particular CCR7expressing T cells.

Monocytes are produced by the bone marrow from haematopoietic stem cellprecursors called monoblasts. Monocytes may differentiate intomacrophages or dendritic cells. Monocytes and their macrophage anddendritic cell progeny serve a number of functions in the immune systemincluding phagocytosis, antigen presentation and cytokine production.Monocytes may be characterized with reference to expression of the cellsurface marker CD14, optionally together with CD16. Classical monocytesmay be characterized by high level expression of the CD14 cell surfacereceptor (CD14++ CD16− monocyte). Non-classical monocytes may becharacterized by low level expression of CD14 and with additionalco-expression of the CD16 receptor (CD14+CD16++ monocyte). Intermediatemonocytes may be characterized by high level expression of CD14 and lowlevel expression of CD16 (CD14++CD16+ monocytes). Macrophages arederived from monocytes and are responsible for protecting tissues fromforeign substances. They are cells that possess a large smooth nucleus,a large area of cytoplasm and internal vesicles for processing foreignmaterial. The term “macrophage” may refer to a monocyte-derived cellexpressing one or more of the following cell surface markers CD14,CD11b, Lysozyme M, MAC-1/MAC-3 and CD68. The term macrophage includesboth activated and un-activated macrophages. Activated macrophages maybe characterized by expression of one or more of CD69, ENG, FCER2 andIL2RA, HLA-DR, CD86. Un-activated macrophages have not yet receivedactivating signals through for example TLR receptors and therefore theyexpress less activation markers on the cell surface which correlateswith lesser maturation. M1 macrophages may be characterized byexpression of one or more of CD16⁺CD32⁺CD64⁺ and secrete mainly IL-23and IL-1, TNF, IL-6 and high levels of IL-12 and in addition effectormolecules such as iNOS and ROI. M1 macrophages have cytotoxic featuresas opposed to M2 macrophages. M2 macrophages may be characterized byexpression of one or more of SRA/B⁺CD163⁺MR⁺CD14⁺ and express TGFβ,IL-10 and IL-1Ra. Tumour associated macrophages (TAMs) share manycharacteristics with the M2 macrophages and are considered as M2polarised macrophages. The M1/M2 paradigm can also be found in monocytesubsets where CD14⁺CD16⁻CXC3R1^(low) monocytes are considered the“inflammatory” subset and the CD14^(low)CD16⁺CXC3R1^(high) are“resident” monocytes.

The three major types of lymphocyte are T cells, B cells and naturalkiller (NK) cells. The term “T-lymphocyte” includes CD4⁺ T cells such asT helper cells (Th1 cells and Th2 cells), and CD8⁺ T cells such ascytotoxic T cells. Th1 cells may be characterized by expression of CCR5and/or by production of IFN-γ. Th2 cells may be characterized byexpression of CCR3 and/or by production of IL-4.

The claimed methods may, in particular, target eosinophils. Eosinophiliais an important component of certain respiratory conditions and may bedefined as the presence of more than 500 eosinophils/microliter ofblood. Thus, reducing numbers of circulating eosinophils represents animportant therapeutic approach. Eosinophils, or eosinophil granulocytes,are white blood cells and represent an important immune systemcomponent. Along with mast cells, they also control mechanismsassociated with allergy and asthma. They are granulocytes that developduring haematopoiesis in the bone marrow before migrating into blood.

The name “eosinophil” derives from the eosinophilic “acid-loving”properties of the cell. Normally transparent, it is this affinity thatcauses them to appear brick-red after staining with eosin, a red dye,using the Romanowsky method. The staining is concentrated in smallgranules within the cellular cytoplasm, which contain many chemicalmediators, such as histamines and proteins such as eosinophilperoxidase, ribonuclease (RNase), deoxyribonucleases, lipase,plasminogen, and major basic protein. These mediators are released by aprocess called degranulation following activation of the eosinophil, andare toxic to both parasite and host tissues.

Eosinophils develop and mature in bone marrow. They differentiate frommyeloid precursor cells in response to the cytokines interleukin 3(IL-3), interleukin 5 (IL-5), and granulocyte macrophagecolony-stimulating factor (GM-CSF). Eosinophils produce and store manysecondary granule proteins prior to their exit from the bone marrow.After maturation, eosinophils circulate in blood and migrate toinflammatory sites in tissues in response to chemokines such as CCL11(eotaxin-1), CCL24 (eotaxin-2), CCL5 (RANTES) and MCP1/4. Eosinophilsmay be activated by Type 2 cytokines released from a specific subset ofhelper T cells (Th2); IL-5, GM-CSF, and IL-3 are important foreosinophil activation as well as maturation. CD44 and CD69 have beenshown to represent different types of cell-surface activation markersfor human eosinophils. CD69 is absent from “fresh” eosinophils butexpressed following activation (using cytokines). CD44 on the other handis constitutively expressed but expression is significantly up-regulatedin response to activation (Matsumoto et al., Am. J. Respir. Cell Mol.Biol., Volume 18, Number 6, June, 1998 860-866). Cell specific markersfor eosinophils include CD9 and CDw125.

CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressed on theseaforementioned cells bind to chemokines such as CCL2 (binds CCR2), CCL3,CCL5, CCL9 (binds CCR1), CCL11 (binds CCR3), CCL12 (binds CCR2), CCL-14(binds CCR1), CCL16 (binds CCR1), CCL28 (binds CCR3), CCL24 (bindsCCR3), CCL26 (binds CCR3) and/or CXCL8 (binds CXCR1 and CXCR2).Chemokines MIP1γ (CCL9), MRP-2 (CCL10), MIp-1δ (CCL15) and CCL23 appearto bind CCR1 only.

Chemokines Eotaxin (CCL11) and CCL24 (Eotaxin-2) only bind CCR3.

Chemokine MIP1β (CCL4) only binds CCR5.

CXCR1 binds CXCL6, CXCL7, CXCL8.

CXCR2 binds CXCL1, 2, 3, 5, 6, 7, 8.

CCR1 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 1. The HGNC ID for this gene is 1602.The gene is located at chromosome position 3p21. The previous symbol andname CMKBR1, SCYAR1. Synonyms for this gene include CD191, CKR-1, MIP1αR. The Entrez Gene reference sequence for CCR1 is 1230 as available on13 Jun. 2011, which is incorporated herein by reference in its entirety.

CCR2 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 2. The HGNC ID for this gene is 1603.The gene is located at chromosome position 3p21. The previous symbol andname for the gene is CMKBR2. Synonyms for this gene include CC-CKR-2,CD192, CKR2, FLJ78302, MCP-1-R. The NCBI Reference Sequence isNM_001123041.2 as available on 13 Jun. 2011, which is incorporatedherein by reference in its entirety.

CCR3 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 3. The HGNC ID for this gene is 1604.The gene is located at chromosome position 3p21.3. The previous symboland name for the gene is CMKBR3. Synonyms for this gene includeCC-CKR-3, CD193 and CKR3. The Genbank reference sequence for CCR3 isAF247361.1 as available on 13 Jun. 2011, which is incorporated herein byreference in its entirety.

CCR5 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 5. The HGNC ID for this gene is 1605.The gene is located at chromosome position 3p21. The previous symbol andname for the gene is CMKBR5. Synonyms for this gene include CC-CKR-5,CD195 CKR-5, IDDM22 and CKR5. The Entrez Gene reference sequence forCCR5 is 1234 as available on 13 Jun. 2011, which is incorporated hereinby reference in its entirety.

CXCR1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) receptor 1. The HGNC ID for thisgene is 6026. The gene is located at chromosome position 2q35. Theprevious symbol and name for the gene is CMKAR1, IL8RA, “interleukin 8receptor, alpha”. Synonyms for this gene include CD181, CDw128a, CKR-1.The Genbank reference sequence for CXCR1 is U11870.1 as available on 13Jun. 2011, which is incorporated herein by reference in its entirety.

CXCR2 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) receptor 2. The HGNC ID for thisgene is 6027. The gene is located at chromosome position 2q35. Theprevious symbol and name for the gene is IL8RB, “interleukin 8 receptor,beta”. Synonyms for this gene include CD182, CMKAR2. The Genbankreference sequence for CXCR2 is U11869.1 as available on 13 Jun. 2011,which is incorporated herein by reference in its entirety.

CCR7 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) receptor 7. The HGNC ID for this gene is 1608.The gene is located at chromosome position 17q12-q21.2. The previoussymbol and name for the gene is CMKBR7, EBI1. Synonyms for this geneinclude BLR2, CD197 and CDw197. The RefSeq reference sequence for CCR7is NM_001838.3 as available on 13 Jun. 2011, which is incorporatedherein by reference in its entirety.

The various embodiments of the methods of the invention may involvespecific binding interactions with any one or more of these furthercell-surface markers in addition to the removal based upon binding toCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7. Suitable bindingreagents can be prepared to specifically bind to these cell-surfacemarkers. The discussion of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/orCCR7 specific binding reagents thus applies mutatis mutandis.

Treatment according to the various embodiments of the invention mayresult in alleviation or amelioration of symptoms, prevention ofprogression, regression of the condition, or complete recovery.Measurable parameters of successful treatment include one or more, up toall, of lungfunction with spirometry, PEF, lung biopsy. Pulsoxymeter.paO2, paCO2. In specific embodiments, a single treatment is sufficientto cause a depletion of around 10%, 20%, 30%, 40%, 50%, 60% or 70%, orhigher up to 80%, 90%, 95% or more, or any range of values between andincluding these amounts, of one or more of a specific chemokinereceptor, in particular one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7, expressing cells from the peripheral blood of thepatient. In specific embodiments, at least around 50% depletion isachieved in a single treatment. Thus, successful treatment may bedefined with reference to depletion of one or more of CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells. Treatment may lead todepletion of between approximately 100 and 500 million CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, such as monocytes, incertain embodiments and more particularly to about 100, 150, 200, 250,300, 350, 400, 450, or 500 million CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7 expressing cells.

By binding to the column through the binding reagent-chemokine receptorinteraction, chemokine receptor expressing cells are immobilized. Theseimmobilized cells express further unoccupied chemokine receptors, whichmay be of the same or different type to those used for capture. Theseadditional chemokine receptors may permit circulating chemokines whichcontribute to the inflammatory condition to be captured from theperipheral blood. Thus, a reduction in circulating (specific) chemokinelevels may provide a measure of successful treatment.

The duration of treatment may be readily determined by one skilled inthe art and will depend upon factors such as the flow rate of theperipheral blood. Duration of treatment may be tied into monitoring ofthe treatment itself, with the treatment considered complete once ameasurable parameter of treatment has reached a defined threshold. Anysuitable parameter may be employed as discussed herein. Thus, forexample, treatment may be considered complete when a reduction in one ormore of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressingcells, such as a 50% reduction in one or more of CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7 expressing cells, has been achieved. Theapheresis system may be operated at a flow rate of around 10-80 mL/min,or more specifically between around 20-70 mL/min, or between around30-60 mL/min. In specific embodiments, the treatment is performed for aperiod of around 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120 etc.,or any range of values between and including these amounts, minutes. Thetreatment is typically not aimed to remove all of the cells expressingthe chemokine receptor in the peripheral blood, as a basal level ofthose cells is required in healthy subjects. However, it has been foundthat only low blood volumes need to be applied to the columns of thevarious embodiments of the invention in order to achieve effectivelevels of depletion of the chemokine receptor-expressing cells. Thus, incertain embodiments, around 10-80% or more specifically around 20, 30,40 or 50%, or any range of values between and including these amounts,of the patient's blood is applied to the column in a single treatment.The volume of blood circulated through the apheresis column or systemmay be in the region of around 1000-3000 ml, such as around 1000, 1200,1400, 1600, 1800 or 2000 ml or any range of values between and includingthese amounts. The treatment may be considered complete once this volumeof blood has been circulated. The patient may be administeredanticoagulants prior to each treatment session. A suitable solution,such as a sterile saline solution, optionally including an anticoagulantsuch as Heparin, may be used for priming the apheresis (extracorporeal)system. An additional volume of anticoagulant may be added to thecircuit at the start of each treatment session, for example as a bolusinjection.

In certain embodiments the invention relies upon a binding reagent whichis capable of specifically binding to a chemokine receptor. Thisspecific binding reaction permits cells expressing the chemokinereceptor to be removed from the peripheral blood of the patient when theblood is passed over the solid support upon or within which the bindingreagent is immobilized. Specific chemokine receptors of interest includeCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7. The binding reagentcan be any binding reagent capable of specifically binding to thereceptor in question. By “specific binding” is meant that the bindingreagent displays sufficient specificity of binding and appropriatebinding affinity/kinetics to permit removal of cells expressing one ormore of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 from theperipheral blood. Whilst it is not precluded that the binding reagent iscapable of binding to other molecules, such as other chemokinereceptors, the binding reagent will preferentially bind to cellsexpressing one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/orCCR7 and in particular to cells expressing increased levels of one ormore of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (as definedfurther herein). The binding reagent capable of specifically binding toCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 may be either anagonist or an antagonist of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/orCCR7, respectively. As the disease condition relies upon up-regulationof expression of or signaling via CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7, in certain embodiments the binding reagent capable ofspecifically binding to CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7is an antagonist of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7,respectively. Chemokines are typically, although not necessarilyexclusively (particularly in the case of truncated or modified forms)agonists of their cognate receptor and serve to activate the cellsexpressing the relevant receptor, as would be appreciated by one skilledin the art.

Antibodies against the relevant chemokine receptor are generallyconsidered to be antagonists, as would be appreciated by one skilled inthe art. Specific examples of binding reagents include proteins orpolypeptides, such as antibodies and receptor ligands, in particularchemokines. The binding reagent may be a nucleic acid molecule incertain embodiments. In some embodiments, the nucleic acid is anaptamer. Nucleic acid aptamers are polynucleotides of approximately15-40 nucleotides long. Nucleic acid aptamers can be made using theSELEX process (systemic evolution of ligands by exponential enrichment)or any other process known to those of skill in the art.

In other embodiments, the binding reagent may be a peptide, and incertain instances, a peptide aptamer. Peptide aptamers are artificialrecognition molecules that consist of a variable peptide sequenceinserted into a constant scaffold protein (Baines I C, Colas P. Peptideaptamers as guides for small molecule drug discovery. Drug Discov Today.2006; 11:334-341, incorporated herein by reference). A number ofmethodologies, such as phage display, ribosome display and yeasttwo-hybrid screening systems are available for screening a library ofpotential peptide-based binding agents. Similarly protein scaffoldsbased on domains such as fibronectins, ankyrin repeats, protein A, SH3domains, lipocalins and ubiquitin can be used as the binding agent.Again a number of technologies such as phage display and ribosomedisplay are available for screening a library of protein—based bindingagents. Similarly, libraries of candidate chemical compounds can bescreened for specific binding to the relevant chemokine receptor usingsuitable screening techniques known in the art, which may be highthroughput screens in certain embodiments. The candidate binding agentmay be immobilized on a solid support and the ability of the agent tospecifically retain cells expressing the chemokine receptor of interestor labelled chemokine receptors determined. A range of cell types may beapplied to the solid supports to confirm specificity of binding, oralternatively a mixed sample (such as peripheral blood) may be appliedto the solid support. Retention of the cell type of interest (expressingthe appropriate chemokine receptor) can be confirmed to identifysuitable binding agents. A range of small-molecule antagonists of CCR-2are discussed by Xia M and Sui Z in Expert Opin Ther Pat. 2009 March;19(3):295-303—Recent developments in CCR2 antagonists, and incorporatedherein by reference.

In the context of the various embodiments of the present invention theterm “chemokine” also comprises biotinylated or otherwise labelledchemokines. The term “chemokine” also comprises modified and truncatedversions of the chemokine and chemokine fragments with the proviso thatthe modified or truncated form retains its ability to bind to itscognate receptor (and thus remains functional in the context of thevarious embodiments of the invention). The chemokine does notnecessarily need to retain biological activity as it is specific bindingaffinity for CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 that isrequired. In certain embodiments, the chemokine lacks biologicalactivity, for example in terms of activation of the (CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7) receptor. Modifications may be made toimprove protein synthesis, for example uniformity of product and yield.As known to those skilled in the art, exemplary modifications maycomprise amino acid additions, substitutions, deletions or othermodifications to one or more amino acids in the chemokine. Modificationsmay comprise substitution of the wild type amino acid with non-naturalamino acids such as norleucine (NLeu) and derivatized amino acids suchas pyroglutamic acid (pyroGlu). Such modifications may be made tominimize side-product formation during storage and use of the columns ofthe various embodiments of the invention. Modifications may be made toimprove labelling, for example inclusion of a polyethylene glycol (PEG)spacer to facilitate biotinylation. The biotinylation and/or conjugationwith fluorochromes or other labelling groups of the chemokine isperformed in a manner which does not substantially affect the receptorbinding capacity. Site specific biotinylation or other labelling ispreferred as non-selective labelling of chemokines may compromisereceptor binding activity. Biotinylation or other labelling is generallypreferred at or towards the C-terminus of the protein as the inventorshave found that modifications in this area are generally well tolerated(in terms of a minimal effect on receptor binding capability).Biotinylation may be carried out site-specifically at any suitable aminoacid. Examples of suitable amino acids include lysine, diaminopropionicacid and ornithine. Generally, reference may be made to Natarajan S etal, Int. J. Pept. Protein Res., 1992, 40, 567-74; Baumeister B, Int. J.Peptide Res. And Therapeutics, 2005, 11, 139-141; Bioconjugatetechniques 2^(nd) edition, Greg T. Hermanson, incorporated by referenceherein in its entirety.

Truncations may involve deletion of either N or C terminal amino acidsas appropriate, or both. Typically, the truncated version will retainthe residues required for the chemokine to fold correctly, for exampleto retain a chemokine fold structure, consistent with the requirementthat a truncated version must retain the ability to bind to the relevantreceptor (expressed by (on the surface of) a leukocyte). Chemokinemolecules typically include disulphide bonds between the 1^(st) and3^(rd) and 2^(nd) and 4^(th) cysteine residues respectively, as would beunderstood by one skilled in the art. Where sequences are presentedherein, it is assumed that these disulphide bonds will form in thefolded protein (unless otherwise stated). Truncated versions maycomprise anywhere between 1 and 100 less amino acids, such as 1, 2, 3,4, 5 etc amino acids, than the wild type amino acid sequence in certainembodiments. Of course, truncated versions may comprise furthermodification as detailed herein. The modified or truncated version mayhave 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or moreoverall amino acid sequence identity with the full length wild typechemokine (where a deletion is counted as a difference in amino acidsequence) in certain embodiments. Over the common sequence between themolecules (i.e the amino acids that have not been deleted), there may be80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acidsequence identity in certain embodiments. Sequence identity may bedetermined using known algorithms, such as BLAST or GAP analysis (GCGProgram) (applying default settings), which are freely available.Chemokines may lack the N-terminal signal peptide which is cleaved offduring synthesis in vivo.

Specific chemokines useful in the various embodiments of the presentinvention for binding to CCR2 include CCL2 (MCP-1), MCP-2, MCP-3, MCP-4(CCL12) and MCP-5. Both MCP-1 and MCP-5 bind solely to the chemokinereceptor CCR2 and so these chemokines may be preferred in someembodiments. Each chemokine is able to bind to a chemokine receptorimplicated in respiratory conditions, in particular sarcoidosis andChronic Obstructive Pulmonary Disease (COPD). More specifically, each ofMCP-1, MCP-2, MCP-3, MCP-4 and MCP-5 are useful for removing CCR2expressing cells from the blood of a patient. Specific chemokines usefulin the various embodiments of the present invention for binding to CCR1,CCR3 and/or CCR5 include CCL5 (RANTES). RANTES is able to bind tochemokine receptors implicated in respiratory conditions, in particularsarcoidosis and Chronic Obstructive Pulmonary Disease (COPD). Morespecifically, RANTES is useful for removing CCR1, CCR3 and/or CCR5expressing cells from the blood of a patient.

Specific chemokines useful for binding to CCR1 include CCL9, MRP-2(CCL10), CCL14, CCL15, CCL16 and CCL23 Specific chemokines useful forbinding to CCR3 include CCL11 (Eotaxin), CCL28, CCL24 (Eotaxin 2) andCCL26. Specific chemokines useful for binding to CCR5 include CCL3,CCL4, CCL5, CCL8 with CCL4 binding only to CCR5. Specific chemokinesuseful in the various embodiments of the present invention for bindingto CXCR1 and/or CXCR2 include CXCL8 (IL-8). IL-8 is able to bind tochemokine receptors implicated in respiratory conditions, in particularsarcoidosis and Chronic Obstructive Pulmonary Disease (COPD). Specificchemokines useful in the various embodiments of the present inventionfor binding to CXCR1 include CXCL6, CXCL7, CXCL8. Specific chemokinesuseful in the various embodiments of the present invention for bindingto CXCR2 include CXCL1, 2, 3, 5, 6, 7, 8. CCL19 is useful for theremoval of CCR7 expressing cells.

The chemokines described in greater detail herein (with reference to therelevant figures and amino acid sequences, as set forth in the SEQ IDNOs) may each be applied according to the present invention.

The modified and truncated chemokines described in greater detail herein(with reference to the relevant amino acid sequences, as set forth inthe SEQ ID NOs and accompanying experimental examples) may each beapplied according to the present invention. Such modified forms mayinstruct the skilled person regarding additional modified forms of thesame and other chemokines which may be suitable for use in theinvention. Chemokines show variable sequence homology varying from lessthan 20% to over 90% but all share very similar tertiary structuresconsisting of a disordered N-terminus, followed by a long loop (theN-loop) that ends in a 3₁₀ helix, a 3-stranded β-sheet and a C-terminalhelix. The overlall topology is stabilsed by disulphide bonds. Thiscommon tertiary structure is a common feature of the chemokine proteinfamily (Fernandez E J and Lolis E., Annu. Rev. Pharmacol. Toxicol., 202,42, 469-99; Allen S J et al, Annu. Rev. Immunol., 2007, 25, 787-820,incorporated herein by reference).

Truncations within this N-terminal region can maintain binding to thereceptor but can lead to a change or loss of function (for examplesZhang Y J et al, J. Biol. Chem., 1994, 269, 15918; Gong J-H andClark-Lewis I., J. Exp. Med., 1995, 181, 631-640; Fernandez E J andLolis E., Annu. Rev. Pharmacol. Toxicol., 202, 42, 469-99; Allen S J etal, Annu. Rev. Immunol., 2007, 25, 787-820, each of which isincorporated herein by reference). Truncations at the C-terminus of thechemokine can also be made and maintain receptor binding activity(Treating Inflammatory Disorders, Ola Winqvist and Graham Cotton,WO2010/029317, incorporated herein by reference in its entirety).

In other embodiments, fragments and variants of chemokines are used inthe devices and methods as disclosed herein. More particularly, suchfragments and variants retain the ability to specifically bind to theircognate chemokine receptor. Chemokines are known by those skilled in theart to share specific receptor binding domains, including a similarmonomeric fold, characterized, for example, by a disorderedamino-terminal domain, followed by a conserved core region, consistingof the so called “N-loop,” three anti-parallel β-strands, and acarboxyl-terminal α-helix. While not being bound by theory, it isbelieved that the chemokine-chemokine receptor interaction is a two-stepmechanism, in which the core of the chemokine interacts first with abinding site formed by the extracellular domains of the receptor, whileanother interaction is formed between the chemokine N terminus and asecond binding site on the receptor in order to trigger receptoractivation. Thus, a “fragment,” such as a functional fragment of achemokine is intended to mean a portion of the amino acid sequence ofthe protein that retains binding for its cognate receptor. The fragmentmay include, for example, the monomeric fold region, or portions thereofsuch as the amino-terminal domain, the conserved core region and/or the“N-loop,” the anti-parallel β-strands, and/or the carboxyl-terminalα-helix or combinations and portions thereof.

Further, it is recognized that a polypeptide can be considerably mutatedwithout materially altering one or more of the polypeptide's functions,for example, without altering specific binding and/or the folding of theprotein. The genetic code is well known to be degenerate, and thusdifferent codons encode the same amino acids. Even where an amino acidsubstitution is introduced, the mutation can be conservative and have nomaterial impact on the essential functions of a protein (see forexample, Stryer, Biochemistry 4th Ed., W. Freeman & Co., New York, N.Y.,1995). This includes, for example, the ability of the protein to bindand interact with other proteins, such as a truncated chemokine bindingto its cognate receptor.

In some examples, part of a polypeptide chain can be deleted withoutimpairing or eliminating all of its functions. For example, the deletionof between about 1 and about 20 amino acids on the C- and/or N-terminus,such as deletions of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 amino acids at the C- and/or N-terminus,can result in a chemokine that retains function, such as specificbinding of its cognate receptor. Such truncations can retain the fullfunction of an entire protein, and/or can allow for retained functionssuch as protein-protein interactions as in the case of ligand-receptorinteractions. Chemokines having deletions of a small number of aminoacids, for example, less than about 20% (such as less than about 18%,less than about 15%, less than about 10%, less than about 8%, less thanabout 5%, less than about 2%, or less than about 1%) of the total numberof amino acids in the wild type chemokine can also be used in themethods and devices disclosed herein. Moreover, insertions or additionscan be made in the polypeptide chain for example, adding epitope tags,without impairing or eliminating its functions (Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publ. Assoc. andWiley-Intersciences, 1998). Other modifications that can be made withoutmaterially impairing one or more functions of a polypeptide include, forexample, in vivo or in vitro chemical and biochemical modifications orthe incorporation of unusual amino acids. In some examples, a functionalfragment of a chemokine may consist of about 10 or more, about 25 ormore, about 50 or more, about 75 or more, about 100 or more, about 125or more, about 150, about 175 or more, or about more or 200 or moreamino acid residues of a chemokine amino acid sequence.

In some examples, the chemokine or a functional fragment thereof has anamino acid that has at least about 60% or 65% sequence identity, about70% or 75% sequence identity, about 80% or 85% sequence identity, about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identityover its full length as compared to a reference sequence, such as thosedetailed herein, for example using the NCBI Blast 2.0 gapped BLAST setto default parameters. Alignment may also be performed manually byinspection. One or more conservative amino acid modifications can alsobe made in the chemokine amino acid sequence, whether an addition,deletion or modification, that does not substantially alter the3-dimensional structure of the polypeptide or its ability to bind to thecognate receptor. For example, a conservative amino acid substitutiondoes not affect the ability of the chemokine to specifically bind itscognate receptor. Conservative substitution tables providingfunctionally similar amino acids are well known in the art. Thefollowing six groups each contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

Peptides, such as chemokines and fragments thereof, can be modified by avariety of chemical techniques to produce derivatives having essentiallythe same activity or function—such as binding to a cognate receptor—asthe unmodified peptides, and optionally having other desirableproperties. For example, carboxylic acid groups of the protein, whethercarboxyl-terminal or side chain, may be provided in the form of a saltof a pharmaceutically-acceptable cation or esterified to form a C1-C16ester, or converted to an amide of formula NR1R2 wherein R1 and R2 areeach independently H or C1-C16 alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the peptide,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C1-C16 alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C1-C16alkoxy or to a C1-C16 ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains can be substituted with one ormore halogen atoms, such as F, Cl, Br or I, or with C1-C16 alkyl, C1-C16alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the peptide side chains can beextended to homologous C2-C4 alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups. Those skilled in the art will also recognize methods forintroducing cyclic structures into the peptides of this disclosure toselect and provide conformational constraints to the structure thatresult in enhanced stability. For example, a C- or N-terminal cysteinecan be added to the peptide, so that when oxidized the peptide willcontain a disulfide bond, generating a cyclic peptide. Other peptidecyclizing methods include the formation of thioethers and carboxyl- andamino-terminal amides and esters.

Peptidomimetic and organomimetic embodiments are also within the scopeof the present disclosure, whereby the three-dimensional arrangement ofthe chemical constituents of such peptido- and organomimetics mimic thethree-dimensional arrangement of the peptide backbone and componentamino acid side chains, resulting in such peptido- and organomimetics ofthe proteins of this disclosure. For computer modeling applications, apharmacophore is an idealized, three-dimensional definition of thestructural requirements for biological activity. Peptido- andorganomimetics can be designed to fit each pharmacophore with currentcomputer modeling software (using computer assisted drug design orCADD). See Walters, “Computer-Assisted Modeling of Drugs”, in Klegerman& Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press:Buffalo Grove, Ill., pp. 165 174 and Principles of Pharmacology Munson(ed.) 1995, Ch. 102, for descriptions of techniques used in CADD. Alsoincluded within the scope of the disclosure are mimetics prepared usingsuch techniques.

Amino acids in a peptide, polypeptide, or protein generally arechemically bound together via amide linkages (CONN). Additionally, aminoacids may be bound together by other chemical bonds. For example,linkages for amino acids or amino acid analogs can include CH2NH—,—CH2S—, —CH2-CH2-CH═CH— (cis and trans), —COCH2-, —CH(OH)CH2-, and—CHH2SO— (These and others can be found in Spatola, in Chemistry andBiochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds.,Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review);Morley, Trends Pharm Sci pp. 463-468, 1980; Hudson, et al., Int J PeptProt Res 14:177-185, 1979; Spatola et al. Life Sci 38:1243-1249, 1986;Harm J. Chem. Soc Perkin Trans. 1307-314, 1982; Almquist et al. J. Med.Chem. 23:1392-1398, 1980; Jennings-White et al. Tetrahedron Lett23:2533, 1982; Holladay et al. Tetrahedron. Lett 24:4401-4404, 1983; andHruby Life Sci 31:189-199, 1982.

CCL2 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 2, also known as MCP-1. The HGNC ID forthis gene is 10618. The gene is located at chromosome position17q11.2-q21.1. The previous symbol and name for the gene is SCYA2 “smallinducible cytokine A2 (monocyte chemotatic protein 1, homologus to mouseSig-je)”. Synonyms for this gene include GDCF-2, HC11, MCP1, MGC9434,SMC-CF, “monocyte chemoattractant protein-1”, “monocyte chemotactic andactivating factor”, “monocyte chemotactic protein 1, homologous to mouseSig-je”, “monocyte secretory protein JE”, “small inducible cytokinesubfamily A (Cys-Cys), member 2”. The Genbank reference sequence forCCL2 is BC009716.1 as available on 13 Jun. 2011, which is incorporatedherein by reference in its entirety.

CCL4 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 4. The HGNC ID for this gene is 10630.The gene is located at chromosome position 17q12-q23. The previoussymbol and name for the gene is LAG1, SCYA4, “small inducible cytokineA4 (homologous to mouse Mip-1b)”. Synonyms for this gene include Act-2,AT744.1, MIP-1-beta. The Genbank reference sequence for CCL4 is M23502.1as available on 13 Jun. 2011, which is incorporated herein by referencein its entirety.

CCL8 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 8, also known as MCP-2. The HGNC ID forthis gene is 10635. The gene is located at chromosome position 17q11.2.The previous symbol and name for the gene is SCYA8, “small induciblecytokine subfamily A (Cys-Cys), member 8 (monocyte chemotactic protein2)”. Another synonym for this gene is HC14. The Genbank referencesequence for CCL8 is X99886.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CCL7 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 7, also known as MCP-3. The HGNC ID forthis gene is 10634. The gene is located at chromosome position17q11.2-q12. The previous symbol and name for the gene is SCYA6, SCYA7,“small inducible cytokine A7 (monocyte chemotactic protein 3)”. Synonymsfor this gene include FIC, MARC, MCP-3, MCP3, NC28, “monocytechemoattractant protein 3”, “monocyte chemotactic protein 3”. TheGenbank reference sequence for CCL7 is AF043338 as available on 13 Jun.2011, which is incorporated herein by reference in its entirety.

CCL13 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 13, also known as MCP-4. TheHGNC ID for this gene is 10611. The gene is located at chromosomeposition 17q11.2. The previous symbol and name for the gene is SCYA13,“small inducible cytokine subfamily A (Cys-Cys), member 13”. Synonymsfor this gene include CKb10, MCP-4, MGC17134, NCC-1, SCYL1. The Genbankreference sequence for CCL13 is AJ001634 as available on 13 Jun. 2011,which is incorporated herein by reference in its entirety.

MCP-5 is a mouse chemokine in the CC chemokine family. It is also knownas Chemokine (C—C motif) ligand 12 (CCL12) and, due to its similaritywith the human chemokine MCP-1, sometimes it is called MCP-1-relatedchemokine. The gene for MCP-5 is found in a cluster of CC chemokines onmouse chromosome 11. The NCBI reference sequence for CCL12 isNC_000077.5. Previous symbol SCYA12 as available on 13 Jun. 2011, whichis incorporated herein by reference in its entirety.

CCL3 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 3, also known as MIP-1a. The HGNC IDfor this gene is 10627. The gene is located at chromosome position17q12. The previous symbol and name for the gene is SCYA3, “smallinducible cytokine A3 (homologous to mouse Mip-1a)”. Synonyms for thisgene include G0S19-1, LD78ALPHA, MIP-1-alpha. The Genbank referencesequence for CCL3 is M23178.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CCL5 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor chemokine (C—C motif) ligand 5, also known as RANTES. The HGNC IDfor this gene is 10632. The gene is located at chromosome position17q11.2-q12. The previous symbol and name for the gene is D17S136E,SCYA5, “small inducible cytokine A5 (RANTES)”. Synonyms for this geneinclude “beta-chemokine RANTES”, MGC17164, RANTES, “regulated uponactivation, normally T-expressed, and presumably secreted”, “SIS-delta”,SISd, “small inducible cytokine subfamily A (Cys-Cys), member 5”,“T-cell specific protein p288”, “T-cell specific RANTES protein”,TCP228. The Genbank reference sequence for CCL5 is AF043341.1 asavailable on 13 Jun. 2011, which is incorporated herein by reference inits entirety.

IL8 is the gene symbol approved by the HUGO Gene Nomenclature Committeefor interleukin 8, also known as CXCL8. The HGNC ID for this gene is6025. The gene is located at chromosome position 4q13-q21. Synonyms forthis gene include 3-10C, AMCF-I, b-ENAP, “chemokine (C—X—C motif) ligand8”, CXCL8, GCP-1, IL-8, K60, LECT, LUCT, LYNAP, MDNCF, MONAP, NAF,NAP-1, SCYB8, TSG-1. The Genbank reference sequence for CXCL8 isY00787.1 as available on 13 Jun. 2011, which is incorporated herein byreference in its entirety.

CCL11 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 11, also known as eotaxin.The HGNC ID for this gene is 10610. The gene is located at chromosomeposition 17q21.1-q21.2. The previous symbol and name for the gene isSCYA11, “small inducible cytokine subfamily A (Cys-Cys), member 11(eotaxin)”. Synonyms for this gene include MGC22554 and “eotaxin-1”. TheGenbank reference sequence for CCL11 is AB063614.1 as available on 13Jun. 2011, which is incorporated herein by reference in its entirety.

CCL15 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 15, also known as HCC-2 andLkn-1. The HGNC ID for this gene is 10613. The gene is located atchromosome position 17q11.2. The previous symbol and name for the geneis SCYA15, “small inducible cytokine subfamily A (Cys-Cys), member 15”.Synonyms for this gene include “CC chemokine 3”, “chemokine CC-2”,HCC-2, HMRP-2B, “leukotactin 1”, Lkn-1, “macrophage inflammatory protein5”, “MIP-1 delta”, MIP-1d, MIP-5, NCC-3, SCYL3. The Genbank referencesequence for CCL15 is AF031587.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CCL14 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 14. The HGNC ID for this geneis 10612. The gene is located at chromosome position 17q11.2. Theprevious symbol and name for the gene is SCYA14, “small induciblecytokine subfamily A (Cys-Cys), member 14”. Synonyms for this geneinclude CKb1, HCC-1, HCC-3, MCIF, NCC-2, SCYL2. The Genbank referencesequence for CCL14 is Z49270.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CCL16 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 16. The HGNC ID for this geneis 10614. The gene is located at chromosome position 17q11.2. Theprevious symbol and name for the gene is SCYA16, “small induciblecytokine subfamily A (Cys-Cys), member 16”. Synonyms for this geneinclude CKb12, HCC-4, LCC-1, LEC, LMC, Mtn-1, NCC-4, SCYL4. The Genbankreference sequence for CCL16 is AB007454.1 as available on 13 Jun. 2011,which is incorporated herein by reference in its entirety.

CCL18 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 18 (pulmonary andactivation-regulated). The HGNC ID for this gene is 10616. The gene islocated at chromosome position 17q11.2. The previous symbol and name forthe gene is SCYA18, “small inducible cytokine subfamily A (Cys-Cys),member 18, pulmonary and activation-regulated”. Synonyms for this geneinclude AMAC-1, CKb7, DC-CK1, DCCK1, MIP-4, PARC. The Genbank referencesequence for CCL18 is Y13710.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CCL23 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 23. The HGNC ID for this geneis 10622. The gene is located at chromosome position 17q11.2. Theprevious symbol and name for the gene is SCYA23, “small induciblecytokine subfamily A (Cys-Cys), member 23”. Synonyms for this geneinclude Ckb-8, CKb8, MIP-3, MPIF-1. The Genbank reference sequence forCCL23 is U58913.1 as available on 13 Jun. 2011, which is incorporatedherein by reference in its entirety.

CCL24 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 24, also known as eotaxin-2and MPIF-2. The HGNC ID for this gene is 10623. The gene is located atchromosome position 7q11.23. The previous symbol and name for the geneis SCYA24, “small inducible cytokine subfamily A (Cys-Cys), member 24”.Synonyms for this gene include “CK-beta-6”, Ckb-6, MPIF-2, MPIF2,“eotaxin-2”, “myeloid progenitor inhibitory factor 2”. The Genbankreference sequence for CCL24 is U85768.1 as available on 13 Jun. 2011,which is incorporated herein by reference in its entirety.

CCL26 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 26, also known as eotaxin-3.The HGNC ID for this gene is 10625. The gene is located at chromosomeposition 7q11.22. The previous symbol and name for the gene is SCYA26,“small inducible cytokine subfamily A (Cys-Cys), member 26”. Synonymsfor this gene include “CC chemokine IMAC”, IMAC, MIP-4a, MIP-4alpha,TSC-1, “chemokine N1,” “eotaxin-3”, “macrophage inflammatory protein4-alpha”, “small inducible cytokine A26”, “thymic stroma chennokine-1”.The Genbank reference sequence for CCL26 is AF124601.1 as available on13 Jun. 2011, which is incorporated herein by reference in its entirety.

CXCL1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) ligand 1 (melanoma growthstimulating activity, alpha). The HGNC ID for this gene is 4602. Thegene is located at chromosome position 4q13.3. The previous symbol andname for the gene is “fibroblast secretory protein”, FSP, GRO1, “GRO1oncogene (melanoma growth stimulating activity, alpha)”, MGSA. Synonymsfor this gene include GROa, MGSA-a, NAP-3, SCYB1. The Genbank referencesequence for CXCL1 is J03561.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CXCL2 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) ligand 2. The HGNC ID for thisgene is 4603. The gene is located at chromosome position 4q13.3. Theprevious symbol and name for the gene is GRO2, “GRO2 oncogene”. Synonymsfor this gene include CINC-2a, GROb, MGSA-b, MIP-2a, SCYB2. The Genbankreference sequence for CXCL2 is M36820.1 as available on 13 Jun. 2011,which is incorporated herein by reference in its entirety.

CXCL3 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) ligand 3. The HGNC ID for thisgene is 4604. The gene is located at chromosome position 4q21. Theprevious symbol and name for the gene is GRO3, “GRO3 oncogene”. Synonymsfor this gene include CINC-2b, GROg, MIP-2b, SCYB3. The Genbankreference sequence for CXCL3 is M36821.1 as available on 13 Jun. 2011,which is incorporated herein by reference in its entirety.

CXCL5 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) ligand 5. The HGNC ID for thisgene is 10642. The gene is located at chromosome position 4q13.3. Theprevious symbol and name for the gene is SCYB5, “small induciblecytokine subfamily B (Cys-X-Cys), member 5 (epithelial-derivedneutrophil-activating peptide 78)”. A synonym for this gene is ENA-78.The Genbank reference sequence for CXCL5 is X78686.1 as available on 13Jun. 2011, which is incorporated herein by reference in its entirety.

CXCL6 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) ligand 6. The HGNC ID for thisgene is 10643. The gene is located at chromosome position 4q13.3. Theprevious symbol and name for the gene is chemokine (C—X—C motif) ligand6 (granulocyte chemotactic protein 2). Synonyms for this gene includeCKA-3, GCP-2. The Genbank reference sequence for CXCL6 is U83303.1 asavailable on 13 Jun. 2011, which is incorporated herein by reference inits entirety.

PPBP is the gene symbol approved by the HUGO Gene Nomenclature Committeefor pro-platelet basic protein (chemokine (C—X—C motif) ligand 7), alsoknown as CXCL7. The HGNC ID for this gene is 9240. The gene is locatedat chromosome position 412-q13. The previous symbol and name for thegene is THBGB1. Synonyms for this gene include b-TG1, Beta-TG,“beta-thromboglobulin”, “connective tissue-activating peptide III”,CTAP3, CTAPIII, CXCL7, LA-PF4, LDGF, MDGF, NAP-2, NAP-2-L1,“neutrophil-activating peptide-2”, PBP, “platelet basic protein”, SCYB7,TGB, TGB1. The Genbank reference sequence for PPBP is M54995.1 asavailable on 13 Jun. 2011, which is incorporated herein by reference inits entirety.

CXCL11 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—X—C motif) ligand 11. The HGNC ID for thisgene is 10638. The gene is located at chromosome position 4q21. Theprevious symbol and name for the gene is SCYB9B, SCYB11, “smallinducible cytokine subfamily B (Cys-X-Cys), member 11”. Synonyms forthis gene include b-R1, H174, I-TAC, IP-9. The Genbank referencesequence for CXCL11 is U66096.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

CCL19 is the gene symbol approved by the HUGO Gene NomenclatureCommittee for chemokine (C—C motif) ligand 19, also known as MIP-3b. TheHGNC ID for this gene is 10617. The gene is located at chromosomeposition 9p13. The previous symbol and name for the gene is SCYA19,“small inducible cytokine subfamily A (Cys-Cys), member 19”. Synonymsfor this gene include “beta chemokine exodus-3”, “CC chemokine ligand19”, “CK beta-11”, CKb11, “EB11-ligand chemokine”, ELC, exodus-3,“macrophage inflammatory protein 3-beta”, MIP-3b. The Genbank referencesequence for CCL19 is AB000887.1 as available on 13 Jun. 2011, which isincorporated herein by reference in its entirety.

Examples of suitable modified chemokines of the various embodiments ofthe invention containing modifications and/or truncations andspecifically adapted for use in the invention are described in detailherein. MCP-1 has been produced with residue 75, which may be a lysine,as the site of biotinylation on the chemokine (numbering based upon themature protein having the amino acid sequence of SEQ ID NO: 2).Biotinylation permits immobilization of MCP-1 on a solid support (via abiotin-avidin interaction). The basic amino acid sequence of MCP-1,including a 23 amino acid leader sequence is set forth as SEQ ID NO: 1.The amino acid sequence of the mature protein is set forth as SEQ ID NO:2. The inventors have determined that chemokines may display improvedbinding properties where the chemokine is biotinylated via a spacergroup. The spacer may prevent the biotin group from impacting on thebinding affinity of the chemokine. Any suitable spacer group may beemployed. Further modifications may provide the molecule withadvantageous properties. The invention also relates to derivatives oftruncated MCP-1 chemokines. The amino acid sequence of the truncatedversion is set forth as SED ID NO: 3.

Accordingly, in certain embodiments the invention also provides amodified MCP-1 chemokine comprising, consisting essentially of orconsisting of the amino acid sequence set forth as SEQ ID NO: 1, SEQ IDNO: 2 or SEQ ID NO: 3 in which one or more of the followingmodifications have been made:

a) the glutamine residue 1 of SEQ ID NO: 2 has been replaced withpyroglutamine

b) the C terminus is produced as an amide derivative (this may beachieved by synthesis on an amide linker)

c) the (C terminal region) residue at position 98 of SEQ ID NO: 1 orposition 75 of SEQ ID NO:2 or position 67 of SEQ ID NO: 3, which may bea lysine or ornithine residue or other residue suitable for labelling,is biotinylated, optionally via a spacer group, in order to permitimmobilization of the chemokine on a solid support; and/or

d) the methionine residue at position 87 of SEQ ID NO: 1 or position 64of SEQ ID NO: 2 or position 56 of SEQ ID NO: 3 has been replaced withnorleucine.

The (C terminal region) amino acid, which may be a lysine residue or afunctional equivalent, at position 98 of SEQ ID NO: 1 or position 75 ofSEQ ID NO:2 or position 67 of SEQ ID NO: 3 may be biotinylated via asuitable spacer group, such as a polyethylene glycol (PEG) spacer group,in order to permit immobilization of the chemokine on a solid support.In specific embodiments, the PEG spacer is 3,6-dioxo aminooctanoic acid.The sequence and biotinylation of the modified MCP-1 chemokines of theinvention are shown in FIGS. 7 to 9 respectively. The modified MCP-1chemokines may be agonists or antagonists of CCR2 activity. They can betested for activity in a suitable assay, such as cell-based assays. Inparticular, agonist and antagonist properties may be determined infunctional cell-based assay on human CCR2 receptor.

MCP-5 only binds CCR2 and should be selective in its removal of CCR2expressing cells. The full length amino acid sequence, including thesignal peptide, is set forth as SEQ ID NO: 4. The amino acid sequence ofN-terminal processed MCP-5 chemokine is 82 amino acids long and is setforth as SEQ ID NO: 5. An amino acid sequence alignment suggests thatMCP-5 has a C-terminal extension when compared to the amino acidsequence of MCP-1. The results of this alignment are shown in FIG. 10.C-terminal truncated versions of MCP-5 can thus be synthesised. Thistruncated version will comprise, consist essentially of or consist ofMCP-5 residues 1-76, set forth as SEQ ID NO: 6.

Accordingly, in certain embodiments the invention also provides amodified MCP-5 chemokine comprising the amino acid sequence set forth asSEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 in which the isoleucineresidue at position 97 of SEQ ID NO: 4 or at position 75 of SEQ ID NO: 5or SEQ ID NO: 6 has been replaced with lysine. In certain embodiments,the modified MCP-5 chemokine comprises, consists essentially of orconsists of the amino acid sequence of SEQ ID NO: 7. The modified MCP-5chemokine may be biotinylated at the lysine (or a functional equivalent)residue at position 97 of SEQ ID NO: 4 or at position 75 of SEQ ID NO: 5or SEQ ID NO: 6. Biotinylation may be via a suitable spacer group.Specific examples of the spacer group include a PEG spacer, optionally3,6-dioxo aminooctanoic acid. In some embodiments, the C terminus isproduced as an amide derivative. This may be achieved by synthesis on anamide linker. In certain embodiments, the modified MCP-5 chemokinecomprises, consists essentially of or consists of the sequence andbiotinylation shown in FIG. 11. The modified MCP-5 chemokine may be anagonist or an antagonist of CCR2 activity. They can be tested foractivity in a suitable assay, such as cell-based assays. In particular,agonist and antagonist properties may be determined in a functionalcell-based assay on human CCR2 receptor.

Eotaxin has been produced with Lys73 as the site of biotinylation on thechemokine (numbering based upon the mature protein having the amino acidsequence of SEQ ID NO: 9). Biotinylation permits immobilization ofeotaxin on a solid support (via a biotin-avidin interaction). The basicamino acid sequence of eotaxin, including a 23 amino acid leadersequence is set forth as SEQ ID NO: 8. The amino acid sequence of themature protein is set forth as SEQ ID NO: 9. The inventors havedetermined that chemokines may display improved binding properties wherethe chemokine is biotinylated via a spacer group. The spacer may preventthe biotin group from impacting on the binding affinity of thechemokine. Any suitable spacer group may be employed. Furthermodifications may provide the molecule with advantageous properties.

Accordingly, in certain embodiments the invention also provides amodified eoxtaxin chemokine comprising the amino acid sequence set forthas SEQ ID NO: 8 or SEQ ID NO: 9 in which one or more of the followingmodifications have been made:

a) the C terminus is produced as an amide derivative

c) the (C terminal region) lysine residue at position 96 of SEQ ID NO: 8or position 73 of SEQ ID NO: 9 is biotinylated, optionally via a spacergroup, in order to permit immobilization of the chemokine on a solidsupport

d) the methionine residue at position 85 of SEQ ID NO: 8 or position 62of SEQ ID NO: 9 has been replaced with norleucine

e) the Lysine at position 96 of SEQ ID NO: 8 or position 73 of SEQ IDNO: 9 s replaced with another amino acid that can enable biotinylationsuch as ornithine

The (C terminal region) lysine residue at position 96 of SEQ ID NO: 8 orposition 73 of SEQ ID NO: 9 may be biotinylated via a suitable spacergroup, such as a polyethylene glycol (PEG) spacer group, in order topermit immobilization of the chemokine on a solid support. In specificembodiments, the PEG spacer is 3,6-dioxo aminooctanoic acid. Thesequence and biotinylation of a modified eotaxin chemokine of theinvention is shown in FIG. 7. The modified eoxtaxin chemokines may beagonists or antagonists of CCR3 activity. They can be tested foractivity in a suitable assay, such as cell-based assays. In particular,agonist and antagonist properties may be determined in aequorinfunctional cell-based assay on human CCR3 receptor.

An example of a chemokine of the various embodiments of the inventioncontaining modifications and specifically adapted for use in theinvention is described in detail herein (see Example 3 below). Themodified CCL8 (MCP-2) corresponds to residues 1 to 76 of the full lengthmature protein (and lacks the N-terminal signal peptide of 23 aminoacids, which is cleaved off) and thus retains the chemokine fold. TheGln at the N-terminus of the protein is subject to pyroGlu formationunder physiological conditions. Thus Gln1 of the sequence may thus besubstituted with pyroglutamine to prevent mixed species of N-terminalGln and pyroGlu being generated (SEQ ID NO: 13). This improves the yieldof synthesis and ensures a homogeneous chemokine preparation throughcolumn manufacture and use. FmocLys(ivDde)-OH is incorporated as residue75 to facilitate site-specific labelling at this position of the protein(SEQ ID NO: 14). The naturally occurring lysine at position 75 ismodified through biotinylation. A PEG spacer may be incorporated betweenthe ε-amino functionality and the biotin (SEQ ID NO: 15).

Thus, in other embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 13:XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLXP

X1=pyroGlu (but may remain as Gln in some embodiments)

X75=an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

Or SEQ ID NO: 15

XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERVVVRDSMKHLDQIFQNLXP

X1=pyroGlu (but may remain as Gln in some embodiments)

X75=K(PEG-Biotin).

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 4 below). Themodified CCL5 (RANTES) corresponds to residues 1 to 68 of the fulllength mature protein (and lacks the N-terminal signal peptide of 23amino acids, which is cleaved off) and thus retains the chemokine fold.The single methionine (Met67) within the sequence is mutated to lysine,to mitigate against oxidation of this residue during the chain assembly(SEQ ID NO: 16). This Met to Lys substitution provides a lysine atposition 67 which can be modified through biotinylation.FmocLys(ivDde)-OH is incorporated as residue 67 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 17).The biotinylated version comprises, consists essentially of or consistsof the amino acid sequence of SEQ ID NO: 18.

Thus, in other embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 18:

SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVC ANPEKKWVREYINSLEXS

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG (e.g. K(Biotin))

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example below). Themodified CCL2 (MCP-1) corresponds to residues 1 to 76 of the full lengthmature protein (and lacks the N-terminal signal peptide of 23 aminoacids, which is cleaved off) and thus retains the chemokine fold (SEQ IDNO: 10). The Gln at the N-terminus of the protein (Gln1) is substitutedwith pyroglutamine to prevent mixed species of N-terminal Gln andpyroGlu being generated. This improves the yield of synthesis andensures a homogeneous chemokine preparation through column manufactureand use. FmocLys(ivDde)-OH is incorporated as residue 75 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 11).A suitable spacer, such as a PEG spacer, may be incorporated between theε-amino functionality and the biotin. The biotinylated versioncomprises, consists essentially of or consists of the amino acidsequence of SEQ ID NO: 12.

Thus, in other embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of:

SEQ ID NO: 10: XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT

X=pyroGlu or Gln

And/or SEQ ID NO: 12: XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPXT

X1=pyroGlu or Gln

X75 is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, optionally K(PEG-Biotin)

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 9 below). Themodified CCL19 (MIP-313) corresponds to residues 1 to 77 of the fulllength mature protein (and lacks the N-terminal signal peptide of 21amino acids, which is cleaved off) and thus retains the chemokine fold.An additional lysine is inserted at the C-terminus, at position 78. Thechemokine may thus comprise, consist essentially of or consist of theamino acid sequence of SEQ ID NO: 19. FmocLys(ivDde)-OH is incorporatedas residue 78 to facilitate site-specific labelling at this position ofthe protein (SEQ ID NO: 20). The s-amino side chain functionality ofLys(78) is modified through biotinylation. The final protein may thuscomprise, consist essentially of or consist of the amino acid sequenceof SEQ ID NO: 21.

Thus, in other embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 19 or 21:

SEQ ID NO: 19 GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated (e.g.K-biotin), optionally via a spacer molecule such as PEG, in particularK(PEG-Biotin)

SEQ ID NO: 21 GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX

X is K(Biotin)

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 3 below). Themodified CXCL8 (IL-8) corresponds to residues 1 to 77 of the full lengthmature protein (and lacks the N-terminal signal peptide of 22 aminoacids, which is cleaved off) and thus retains the chemokine fold. Anamino acid residue capable of biotinylation, such as lysine orornithine, is added as residue 78 (SEQ ID NO: 22). FmocLys(ivDde)-OH maybe incorporated as residue 78 to facilitate site-specific labelling atthis position of the protein (SEQ ID NO: 23). The additional amino acid,in particular lysine or ornithine, at position 78 is modified throughbiotinylation. A suitable spacer, such as a PEG spacer, may beincorporated between the ε-amino functionality and the biotin (SEQ IDNO: 24).

Thus, in other embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 22 or 24:

SEQ ID NO: 22 AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSX

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG

SEQ ID NO: 24 AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSK(PEG-Biotin)

A further example of a chemokine of the various embodiments of theinvention containing truncations and modifications and specificallyadapted for use in the invention is described in detail herein (seeExample 3 below). The modified CXCL8 (IL-8) corresponds to residues 6 to77 of the full length mature protein, with the first 5 N-terminal aminoacids removed, (and lacks the N-terminal signal peptide of 22 aminoacids, which is cleaved off) and thus retains the chemokine fold. Anamino acid residue capable of biotinylation, such as lysine orornithine, is added as residue 78 (SEQ ID NO: 25). FmocLys(ivDde)-OH maybe incorporated as residue 78 to facilitate site-specific labelling atthis position of the protein (SEQ ID NO: 26). The additional amino acid,in particular lysine or ornithine, at position 78 is modified throughbiotinylation. A suitable spacer, such as a PEG spacer, may beincorporated between the ε-amino functionality and the biotin (SEQ IDNO: 27).

Thus, in other embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 25 or 27:

SEQ ID NO: 25 SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSX

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG

SEQ ID NO: 27 SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSX

X is K(PEG-Biotin)

A further example of a chemokine of the various embodiments of theinvention containing modifications and specifically adapted for use inthe invention is described in detail herein (see Example 9 below). Themodified CCL11 (Eotaxin) corresponds to residues 1 to 74 of the fulllength mature protein (and lacks the N-terminal signal peptide of 23amino acids, which is cleaved off) and thus retains the chemokine fold(SEQ ID NO: 28). The lysine at position 73 may be modified throughbiotinylation. FmocLys(ivDde)-OH is incorporated as residue 73 tofacilitate site-specific labelling at this position of the protein (SEQID NO: 29). A suitable spacer, such as a PEG spacer, may be incorporatedbetween the ε-amino functionality and the biotin. The biotinylatedversion comprises, consists essentially of or consists of the amino acidsequence of SEQ ID NO: 30.

Thus, in other embodiments the invention also relates to a modifiedchemokine comprising, consisting essentially of or consisting of theamino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 30:

SEQ ID NO: 28 GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

SEQ ID NO: 30 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP-NH₂

X is K(PEG-Biotin)

Chemokines useful in the various embodiments of the invention may besynthesised through any suitable means known in the art. Preferably, thechemokines are chemically synthesised as this facilitates modificationand labelling etc. However, recombinant DNA based approaches may also beemployed in combination with appropriate labelling and modificationtechnologies as required. Thus, in certain embodiments the inventionalso provides a nucleic acid molecule encoding the chemokines of thevarious embodiments of the invention. In certain embodiments theinvention also relates to a vector containing such a nucleic acidmolecule and a host cell containing the vector. The vector mayadditionally comprise a suitable promoter operably linked to the nucleicacid molecule, to facilitate transcription of the corresponding mRNAmolecule. The host cell may be capable of expressing the protein bytranscription and translation of the nucleic acid molecule encoding achemokine of the various embodiments of the invention.

The chemokines useful in the various embodiments of the invention can bebiotinylated by methods known in the art such as described in WO00/50088 A2, which is incorporated herein by reference in its entirety.As indicated above, site-specific labelling of the chemokines of thevarious embodiments of the invention is preferable, although anylabelling technique which does not significantly affect thereceptor-binding capacity of the chemokine may be employed. Varioussite-specifically biotinylated chemokines and native chemokines areavailable commercially, for instance from Almac, Craigavon, UK. Inspecific embodiments the one or more chemokines are biotinylated via aspacer group. The spacer may be employed to prevent the biotin groupfrom impacting on the activity of the chemokine, in particular bindingof the chemokine to its cognate receptor. Any suitable spacer thatfacilitates retention of receptor binding properties of the chemokinemay be employed in the various embodiments of the invention. Thus, inthe specific embodiments described above, spacers other than PEG spacersmay be employed as appropriate. In specific embodiments, the spacer is apolyethylene glycol (PEG) spacer. PEG has been shown to be an effectivespacer permitting attachment of biotin to the chemokine (which can thenbe immobilized on the solid support through interaction withstreptavidin) without compromising receptor binding capability.

In the context of the various embodiments of the present invention theterm “antibody” includes all immunoglobulins or immunoglobulin-likemolecules with specific binding affinity for the relevant chemokinereceptor (including by way of example and without limitation, IgA, IgD,IgE, IgG and IgM, combinations thereof, and similar molecules producedduring an immune response in any vertebrate, for example, in mammalssuch as humans, goats, rabbits and mice). Specific immunoglobulinsuseful in the various embodiments of the invention include IgG isotypes.The antibodies useful in the various embodiments of the invention may bemonoclonal or polyclonal in origin, but are typically monoclonalantibodies. Antibodies may be human antibodies, non-human antibodies, orhumanized versions of non-human antibodies, or chimeric antibodies.Various techniques for antibody humanization are well established andany suitable technique may be employed. The term “antibody” also refersto a polypeptide ligand comprising at least a light chain or heavy chainimmunoglobulin variable region which specifically recognizes and bindsan epitope of an antigen, and it extends to all antibody derivatives andfragments that retain the ability to specifically bind to the relevantchemokine receptor. These derivative and fragments may include Fabfragments, F(ab′)₂ fragments, Fv fragments, single chain antibodies,single domain antibodies, Fc fragments etc. The term antibodyencompasses antibodies comprised of both heavy and light chains, butalso heavy chain (only) antibodies. In specific embodiments, theantibodies may be engineered so as to be specific for more than onechemokine receptor, for example bi-specific to permit binding to twodifferent chemokine receptors. Suitable commercially availableantibodies which bind to the chemokine receptors of interest are listedin table 1 below. They may or may not be labelled. General reference maybe made to “Antibodies a laboratory manual: By E Harlow and D Lane. pp726. Cold Spring Harbor Laboratory. 1988”, which reference isincorporated herein in its entirety.

TABLE 1 Commercially available fluorophore labelled antibodies againstspecific chemokine receptors Antibody Fluorophore Supplier CCR2 PerCPCy5.5 Biolegend CCR1 Alexa Fluor 647 Biolegend CCR3 PE Biolegend CCR5 PEBiolegend CXCR1 APC Biolegend CXCR2 PE Biolegend CCR7 PerCP Cy5.5Biolegend

Anti-CCR2 antibodies are described for example in WO 2010/021697,incorporated herein by reference. Further examples of potentially usefulantibodies include MLN-1202, an anti-CCR2 monoclonal antibody currentlyundergoing clinical trials (Millennium Pharmaceuticals).

The chemokine receptor expressing cells may thus be targeted usingalternative binding agents, such as antibodies or other chemicalcompounds, as defined herein, rather than the natural chemokine bindingpartner. This approach is a new approach to treating inflammatoryconditions.

Accordingly, in certain embodiments the invention also provides anapheresis column loaded with a solid support comprising a bindingreagent capable of specifically binding to a chemokine receptorimmobilized directly or indirectly on the support to permit removal of acell expressing the chemokine receptor from the peripheral blood of apatient, wherein the binding reagent is not a chemokine. The bindingreagent capable of specifically binding to the chemokine receptor may bean agonist or an antagonist of the chemokine receptor. In specificembodiments, the binding reagent capable of specifically binding to thechemokine receptor is selected from an antibody and a chemical compound.

In other embodiments the invention thus also provides a method fortreating an inflammatory condition comprising applying peripheral bloodfrom a patient/subject to an apheresis column as defined above (anapheresis column loaded with a solid support comprising a bindingreagent capable of specifically binding to a chemokine receptorimmobilized directly or indirectly on the support to permit removal of acell expressing the chemokine receptor from the peripheral blood of apatient, wherein the binding reagent is not a chemokine) thus removingchemokine receptor expressing cells from the peripheral blood of thepatient/subject. The method may comprise returning the blood depleted ofthe chemokine receptor expressing cells to the patient/subject.

Similarly, in other embodiments the invention provides a binding reagentcapable of specifically binding to a chemokine receptor for use in thetreatment of an inflammatory condition, wherein the binding reagent isimmobilized on a solid support contained within an apheresis column asdefined above (an apheresis column loaded with a solid supportcomprising a binding reagent capable of specifically binding to achemokine receptor immobilized directly or indirectly on the support topermit removal of a cell expressing the chemokine receptor from theperipheral blood of a patient/subject, wherein the binding reagent isnot a chemokine), to which is applied peripheral blood from a patientthus removing chemokine receptor expressing cells from the peripheralblood of the patient.

These aspects of the various embodiments of the invention may beintegrated into the more focused therapeutic aspects of the variousembodiments of the invention (i.e. treating respiratory conditions suchas sarcoidosis and COPD and various aspects thereof) and thus, theremainder of the disclosure, including all specific embodiments appliesmutatis mutandis.

Solid support materials for immobilizing the binding reagents of thevarious embodiments of the invention are known in the art. “Solidsupport” refers to, for example, materials having a rigid or semi-rigidsurface or surfaces, and may take the form of beads, resins, gels,microspheres, or other geometric configurations. A useful supportmaterial is one that does not activate blood cells so as to make themcoagulate or adhere to the support as peripheral whole blood is appliedto the device. In certain embodiments, a support treated with an agentto provide it with anti-coagulation properties, in particular aheparinized support is employed. Alternatively, the blood of the patientmay be treated with an anti-coagulant such as heparin prior toapplication to the support. Useful support materials comprise highmolecular weight carbohydrates, in particular carbohydrates having amolecular weight of 100 kDa or more, such as agarose, in particulateform, optionally cross-linked, and cellulose. Other preferred supportmaterials are polymers, such as carboxylated polystyrene, and glass. Thesupport of the various embodiments of the invention may be provided inthe form of particles or fibres. The support particles may have regularform, such as spheres or beads, or irregular form. They may be porous ornon-porous. A preferred average particle size of the support is from 50μm to 2 mm. In certain embodiments Sepharose™, a cross linked,beaded-form of agarose, is used as column matrix. It is chosen for itsoptimal distribution capacity and can provide a large available area foraffinity binding. Solid supports may be provided in the form of magneticbeads, with the specific binding reagent immobilized on the magneticbeads. Following capture of the (CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7) chemokine receptor expressing cells from the blood, thebeads can be removed from the blood with the aid of an appropriatemagnetic separator.

Methods for immobilizing binding reagents on a solid support are knownin the art. A binding reagent, such as a chemokine, antibody, peptide,nucleic acid or chemical compound, can be immobilized on the support ina direct or indirect manner. Immobilization can be by means of asuitable linker in some embodiments. A preferred method of indirectimmobilization of a binding reagent, such as a chemokine, relies uponthe interaction between biotin and avidin molecules. “Avidin” or “avidinmolecule” refers to any type of protein that specifically binds biotinto the substantial exclusion of other (small) molecules that might bepresent in a biological sample. Examples of avidin include avidins thatare naturally present in egg white, oilseed protein (e.g., soybeanmeal), and grain (e.g., corn/maize), and streptavidin, which is aprotein of bacterial origin Thus, biotinylation of the binding reagentand use of an avidin molecule such as streptavidin immobilized on thesolid support allows reliable attachment of the binding reagent to thesolid support according to methods known in the art. Specifically, sucha method may comprise providing the binding reagent in biotinylatedform, providing a solid support having streptavidin immobilized on itssurface, contacting the support with an aqueous solution of thebiotinylated binding reagent, and rinsing the support with an aqueoussolvent. In addition, binding pair interactions, such asantibody—antigen interactions, may also be utilised for indirectimmobilisation of binding reagent onto a support. In such embodimentsthe support may be derivatised with one member of a binding pair, suchas an antibody or fragment or derivative thereof, as defined herein,which has known affinity for a particular peptide sequence or smallmolecule hapten. Incorporating the other member of the binding pair,such as an antigen, peptide sequence or the hapten onto or into thebinding reagent facilitates immobilisation onto a solid support coatedwith the corresponding antibody or fragment or derivative thereof. Thus,the binding reagent may be modified to include the peptide sequence orhapten into the linear molecule or may be added as a side chain orlabel. Any suitable antibody-antigen pair may be employed. The antibodyfragment or derivative may be any fragment or derivative that retainsspecific binding affinity for the appropriate antigen. Examples includeFab, F(ab′)₂ fragments, scFV, VH domains, single domain antibodies (suchas nanobodies), heavy chain antibodies and humanized version ofnon-human antibodies etc. Other high affinity interactions can beutilised for immobilisation of binding reagents, as long as the bindingreagent can be synthesised or derivatised with one of the interactingpartners and the solid support synthesised or derivatised with the otherinteracting partner without loss of binding activity (i.e. binding ofthe binding reagent to the appropriate chemokine receptor).Immobilization may occur via essentially the same interaction in reversein some embodiments. Thus, the binding reagent which may be a chemokinefor example, may be attached to an antibody as defined herein, and thesolid support derivatised with the antigen. The chemokine may beproduced as a fusion protein with the antibody.

Alternatively binding reagents, such as chemokines and antibodies, canbe immobilised directly onto a solid support using bioconjugationtechniques established in the field. For example direct immobilisationof proteins onto cyanogen bromide activated solid supports via aminofunctionalities within the primary sequence of the protein.Alternatively, sulphydryl functionalities within proteins can be used todirectly immobilise the protein to alkyl halide derivatised supports orsupports containing free thiol functionalities. In further embodiments,proteins containing α-thioester functionalities can be directlyimmobilised on supports containing 1,2 amino thiol moieties (egN-terminal cysteine) using the native chemical ligation reaction.Alternatively proteins modified with ketones and aldehydes can beimmobilised on solid supports derivatised with hydrazinyl, hydrazide andaminoxy functionalities using hydrazone/oxime bond forming ligationreactions (and vice versa). Alternatively ‘Click’ chemistry can be usedto immobilise proteins onto solid supports, whereby the protein and thesupport are derivatised with the appropriate mutually reactive chemicalfunctionalities (azides and alkynes). In other embodiments Staudingerligation chemistry can be used to immobilise appropriately derivatisedproteins onto the appropriately derivatised solid supports.

The solid support is contained within or carried by the apheresiscolumn. Thus, by “loaded” is meant that the column carries or containsthe solid support in a manner such that (peripheral) blood can flowthrough the column in contact with the solid support. Thus, the solidsupport provides a matrix within the column through which blood flows,in continuous fashion in certain embodiments. This permits cellsexpressing the specific chemokine receptor to be removed from the bloodpassing through the column, such that blood exiting the column isdepleted of the specific chemokine receptor-expressing cells. Inspecific embodiments, the apheresis column is loaded with a supportcomprising streptavidin immobilized on the support and one or morebiotinylated binding reagents, such as chemokines, bound to thestreptavidin on the support. The solid support may be comprised of ahigh-molecular weight carbohydrate, optionally cross-linked, such asagarose.

As discussed above, the binding reagent is coupled to the solid support.The relative amounts of binding reagent may be controlled to ensure thatcoupling between the solid support and the binding reagent will beimmediate, minimising the risk of binding reagent decoupling from thesolid support. Thus, it may be ensured that there is a relative excessof immobilization sites for the binding reagent on the solid support.Alternatively, or additionally, following immobilization of the bindingreagent on the solid support, the solid support may be washed to removeany unbound binding reagent.

The apheresis column utilised in the various embodiments of the presentinvention acts as a leukapheresis treatment for respiratory conditions,in particular sarcoidosis and Chronic Obstructive Pulmonary Disease(COPD). The column acts to specifically remove one or more of CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7-expressing monocytes orleukocytes by exploiting the interaction between CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7 expressed on the cell surface and a specificbinding reagent immobilized on a solid support contained within orcarried by the column. The overall column typically comprises, consistsof, or consists essentially of three combined components; 1) a housingwhich contains or carries 2) the solid support and 3) one or morebinding reagents (immobilized thereon) which specifically bind one ormore chemokine receptors. The housing may be manufactured from anysuitable material for clinical use. In certain embodiments the housingis composed of a plastic material. The housing includes an in flow sitefor entry of blood and an out flow site for blood (depleted of targetcells) to exit the column. The housing may be designed to maintain acontinuous blood flow through the solid support matrix. The housing (asshown for example in FIG. 3) may include a top portion which comprises adistribution plate (2) at the inflow site (1) to spread the blood evenlyover the entire matrix area. The distribution plate may act as a firstsafety barrier preventing larger particles flowing through the columnand into the patient. However, the distribution plate is not essentialand may be removed in some embodiments to decrease the overallresistance in the system. The column may contain one or more safetyfilter units (3 and 4) placed at the inflow (1) and/or outflow (5) sitesof the plastic housing. Such filter units may act to prevent particleslarger than blood cells passing in and/or out of the column. The safetyfilter units may contain a plurality of filters, such as two, three orfour filters designed to be a robust barrier and stop all particleslarger than blood cells passing through the column. Inclusion of safetyfilters (3 and 4) at both ends of the column serves to minimize the riskof leakage of particles into the patient, including in the event thatthe device is incorrectly connected resulting in blood flow in theopposite direction to that intended. The safety filters may comprise ofany suitable pore size to prevent particles larger than blood cells frompassing through the column, as would be readily understood by oneskilled in the art. Suitable filters are commercially available. Inspecific embodiments, the pore size of the filter(s) is betweenapproximately 60 μm and 100 μm, more specifically approximately 80 μm.The solid support and binding reagent components are discussed infurther detail herein.

The volume of the housing may be varied depending upon the blood volumesintended to pass through the column. Typically, the volume of thehousing is between approximately 40 ml and 200 ml, more specifically 50ml to 150 ml or 60 ml to 120 ml.

The column is generally applied in the form of an apheresis circuit. Inthis context, the overall system includes the apheresis column, tubingand an appropriate pump to pump the blood around the circuit. The systemis illustrated in FIG. 4. The patient (1) is connected to theextracorporeal circuit via sterile needles to veins in the right and theleft arms. A saline bag (3) is also connected and the saline solution ispumped with a suitable pump (2). Blood is drawn from one arm of thepatient through the sterile tubing system by the blood pump (4) andpassed through the column (6) and back to the patient. The tubing systemmay be connected to the column via any suitable coupling, such asstandard dialysis luer-lock couplings. The couplings on the column maybe colour-coded for correct assembly. For example, red tubing for inflowto the red column top and blue tubing for outflow back to the patient.An air detector (8) may be present in the circuit. Inlet pressure (5)and/or Pven sensors (7) may additionally be employed to monitor thepressure in the circuit.

An apheresis pump, such as the 4008 ADS pump manufactured by FreseniusMedical Care or the Adamonitor pump, may monitor the patient's inflowand outflow. The pump may also monitor the pressure in theextracorporeal circulation. The pump may be able to discriminate air bya bubble catcher and air detector. A clot catcher filter may bepositioned inside the bubble catcher. The pump may also incorporate anoptical detector to distinguish between light, e.g. saline solution orair present in the tubing system and dark e.g. blood present in thetubing system.

A schematic diagram of a suitable pump, showing the air detector andoptical filter is shown in FIG. 8. If the pump system detects airbubbles and optical fluctuations or if extracorporeal pressure valuesare out of the set range, then the pump may stop immediately.Alternatively or additionally a visual/audible alarm may be emitted.

The treatment methods of the various embodiments of the invention maythus rely upon an extracorporeal circuit. The methods may be consideredas ex vivo or in vitro methods and be defined solely with reference tosteps performed outside of the patient. In some embodiments, however,the method further comprises, prior to application of the blood to thecolumn, collecting peripheral blood from the patient. In a furtherembodiment, the method further comprises, following the application ofthe blood to the column, infusing the blood depleted of (CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7) chemokine receptor expressingcells to the patient. This is then a complete leukapheresis treatmentmethod. Thus, a leukaphereis method, for treating respiratoryconditions, in particular sarcoidosis and Chronic Obstructive PulmonaryDisease (COPD), comprises collecting peripheral blood from the patient;applying the peripheral blood to an apheresis column loaded with a solidsupport comprising one or more binding reagents capable of specificallybinding to one or more chemokine receptors, in particular the chemokinereceptor CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7, immobilizeddirectly or indirectly on the support thus removing one or more of CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells from theperipheral blood of the patient; and infusing the depleted blood (ofchemokine receptor expressing cells) to the patient.

The peripheral blood may be continuously collected from the patient.Similarly, the depleted blood may be continuously infused to thepatient, through use of an appropriate circuit as described herein.Thus, the support may be disposed in a column through which the blood ismade to flow. This may be achieved using a suitable pump for example, asalso described herein. Blood flow through the column enables the bindingreagent(s) immobilized on the solid support to capture the cellsexpressing the chemokine receptor, thus depleting them from the bloodand preventing their contribution to the inflammatory respiratoryconditions, in particular sarcoidosis and Chronic Obstructive PulmonaryDisease (COPD).

The methods of the various embodiments of the invention and bindingreagents for use in the methods of the various embodiments of theinvention may require that the patient has been selected for treatmenton the basis of detecting an increase in the level of chemokinereceptor, in particular, one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 expressing cells in a sample obtained from thepatient. Such companion diagnostic methods are described in greaterdetail herein and are based, for example, on the observation that CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression may be elevated inpatients with respiratory conditions, in particular sarcoidosis andChronic Obstructive Pulmonary Disease (COPD). It is shown herein thatsubjects suffering from respiratory conditions such as sarcoidosisexhibit increased frequency of chemokine receptor expressing cells inthe peripheral blood. Subjects with sarcoidosis exhibit increasedfrequency of CCR1 expressing cells such as CCR1 expressing monocytes,compared to healthy controls. Similarly, it is shown herein that themonocytes also express CCR2. It is also shown herein that subjectssuffering from respiratory conditions such as sarcoidosis exhibitincreased frequency of CCR7 expressing cells such as CCR7 expressinglymphocytes, and also central memory T cells, compared to healthycontrols.

Thus, (in this context) in certain embodiments the invention alsoprovides a method of diagnosing, monitoring progression of, ormonitoring treatment of respiratory conditions, in particularsarcoidosis and Chronic Obstructive Pulmonary Disease (COPD) comprisingdetermining:

a) the levels of one or more of the chemokine receptor CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells

b) levels of expression of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7; and/or

c) levels of cells with high expression of one or more of CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 in a sample obtained from asubject, wherein high levels of one or more of CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7 expressing cells, high levels of expression ofone or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 or highlevels of cells with high expression of one or more of CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 or increased levels of one or more ofCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cellscompared to control, increased levels of expression of one or more ofCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 compared to a controlor increased levels of cells with high expression of one or more ofCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 compared to a controlindicate the presence or progression of respiratory conditions, inparticular sarcoidosis and Chronic Obstructive Pulmonary Disease (COPD).Levels of chemokine receptor expression, as opposed to cell numbers, mayalso be investigated as increased levels of chemokine receptorexpression per cell may also be diagnostically relevant. In specificembodiments, the cells relevant to diagnosis etc. of respiratoryconditions such as sarcoidosis comprise monocytes, in particular CCR1and/or CCR2 expressing monocytes. In other embodiments cells relevant todiagnosis etc. of respiratory conditions such as sarcoidosis compriselymphocytes, specifically T lymphocytes such as central memory T cells,in particular CCR7 expressing T cells.

“Diagnosing” is defined herein to include screening for adisease/condition or pre-indication of a disease/condition, identifyinga disease/condition or pre-indication of a disease/condition andchecking for recurrence of disease/condition following treatment. Themethods of the various embodiments of the invention may also haveprognostic value, and this is included within the definition of the term“diagnosis”. The prognostic value of the methods of the variousembodiments of the invention may be used as a marker of potentialsusceptibility to respiratory conditions, in particular sarcoidosis andChronic Obstructive Pulmonary Disease (COPD) by identifying levels ofone or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expression linked to the respiratory condition. Thus patients at riskmay be identified before the disease has a chance to manifest itself interms of symptoms identifiable in the patient. In certain embodiments,diagnosis may be made in conjunction with other objective indicators ofrespiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD): The diagnosis of sarcoidosis maybe made based upon clionical findings; x-ray, biopsy, s-Ca, ACEactivity. COPD diagnosis may also be made by clinical findings; X-rayspirometry and blood gas analysis.

“Monitoring progression of” includes performing the methods to monitorthe stage and/or the state and progression of the respiratoryconditions, in particular sarcoidosis and Chronic Obstructive PulmonaryDisease (COPD). Monitoring progression may involve performing thediagnostic methods multiple times on the same patient to determinewhether the levels of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 expressing cells are increasing, decreasing orremaining stable over a certain time period. This may be in the contextof a treatment regime.

“Monitoring the success of a particular treatment” is defined to includedetermining the levels of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 expressing cells before and after a treatment. Thetreatment is generally one aimed at treating respiratory conditions, inparticular sarcoidosis and Chronic Obstructive Pulmonary Disease (COPD)and may be a treatment according to one of the methods of the variousembodiments of the invention as defined herein. Successful treatment maybe determined with reference to a decrease in one or more of CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells as a result of, orfollowing, the treatment. Thus, in such methods a level of one or moreof CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells isdetermined prior to treatment. This level is recorded and a furtherassessment made at a predetermined time following the treatment. Thecomparison of levels of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 expressing cells permits the success of the treatmentto be monitored. In specific embodiments, a single treatment issufficient to cause a depletion of around 10%, 20%, 30%, 40%, 50%, 60%or 70%, or higher, up to 80%, 90%, 95% or more, or any range of valuesbetween and including these amounts, of one or more specific chemokinereceptors, in particular one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7, expressing cells from the peripheral blood of thepatient. In specific embodiments, at least around 50% depletion isachieved in a single treatment. Thus, successful treatment may bedefined with reference to depletion of one or more of CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells. Treatment may lead todepletion of between approximately 100 and 500 million of one or more ofCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, suchas monocytes, in certain embodiments. Additional factors may be includedto determine successful treatment. For example, a lack of increase inone or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expressing cells following treatment may indicate successful treatmentin terms of preventing further progression of the condition, optionallycombined with an improvement in other markers or staging of therespiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD).

By binding to the column through the binding reagent-chemokine receptorinteraction, chemokine receptor expressing cells are immobilized. Theseimmobilized cells express further unoccupied chemokine receptors, whichmay be of the same or different type to those used for capture. Theseadditional chemokine receptors may permit circulating chemokines whichcontribute to the inflammatory condition to be captured from theperipheral blood. Thus, a reduction in circulating (specific) chemokinelevels may provide a measure of successful treatment. In specificembodiments, the cells relevant to diagnosis etc. of respiratoryconditions such as sarcoidosis comprise monocytes, in particular CCR1and/or CCR2 expressing monocytes. In other embodiments cells relevant todiagnosis etc. of respiratory conditions such as sarcoidosis compriselymphocytes, specifically T lymphocytes such as central memory T cells,in particular CCR7 expressing T cells.

In specific embodiments, the respiratory conditions are selected fromsarcoidosis and Chronic Obstructive Pulmonary Disease (COPD).

The sample in which one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7 expressing cell levels, levels of expression of one or moreof CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 and/or levels ofcells with high expression of one or more of CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7 (defined as CCR2^(hi), CCR1^(hi), CCR3^(hi),CCR5^(hi), CXCR1^(hi), CXCR2^(hi) or CCR7^(hi)) are determined maycomprise any suitable tissue sample or body fluid sample. Generally, thetest sample is obtained from a human subject. Typically, the sample is ablood sample, in particular a peripheral blood sample. The sample maycomprise an adipose tissue biopsy in certain embodiments. The methodsmay involve determining levels of one or more of CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7 expressing monocytes, macrophages orlymphocytes in certain embodiments. In specific embodiments, the cellsrelevant to diagnosis etc. of respiratory conditions such as sarcoidosiscomprise monocytes, in particular CCR1 and/or CCR2 expressing monocytes.In other embodiments cells relevant to diagnosis etc. of respiratoryconditions such as sarcoidosis comprise lymphocytes, specifically Tlymphocytes such as central memory T cells, in particular CCR7expressing T cells.

Levels of CCR2, CCR1, CCR3 or CCR5 expressing cells, levels ofexpression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 and/orlevels of cells with high expression of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 (defined as CCR2^(hi), CCR1^(hi), CCR3^(hi),CCR5^(hi), CXCR1^(hi), CXCR2^(hi) and/or CCR7^(hi)) may be determinedaccording to any suitable method. For example, flow cytometry may beemployed in order to determine the number of cells expressing CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 in the sample, to determinelevels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressionand/or to identify levels of CCR2^(hi), CCR1^(hi), CCR3^(hi), CCR5^(hi),CXCR1^(hi), CXCR2^(hi) and/or CCR7^(hi) cells. Flow cytometrictechniques are described herein and examples of commercially availableantibodies suitably labelled for use in flow cytometry are set out inTable 1 for example. Alternatively, the method may involve steps ofcollecting and fixing the cells in the sample, followed by incubationwith a suitable binding reagent that binds specifically to the CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 chemokine receptor expressingcells in the sample. Any suitable binding reagent, as defined herein,may be employed. For example, a CCR2, CCR1, CCR3, CCR5, CXCR1 or CXCR2specific antibody may be employed. A wash step may be adopted followingan incubation period to remove any unbound reagent. Suitable wash stepsand incubation conditions would be well known to one skilled in the art.The binding reagent may be directly labeled in order to permit antibodybinding to be directly determined. Alternatively a secondary bindingreagent, such as an antibody, may be employed which binds to the firstbinding reagent and carries a label. Again, suitable incubationconditions and wash steps would be apparent to one skilled in the art.The primary and secondary binding reagents may form two halves of abinding pair. The binding interaction should not prevent the primarybinding reagent binding to the CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7 receptor expressing cells, unless a competition assay isbeing employed. The two halves of a binding pair may comprise anantigen-antibody, antibody-antibody, receptor-ligand,biotin-streptavidin pair etc. in certain embodiments. Other techniquesused to quantify chemokine (CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/orCCR7) receptor expressing cell levels, to quantify levels of CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression and/or to quantifylevels of CCR2^(hi), CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi),CXCR2^(hi) and/or CCR7^(hi) cells include PCR-based techniques such asQT-PCR and protein based methods such as western blot. Quantitation maybe achieved with reference to fixed cell lines carrying known numbers ofvarious receptor expressing cells and/or known levels of receptorexpression per cell. Such fixed cell lines are available commercially(for example ChemiScreen™ cell lines from Millipore). Methods analogousto the treatment methods of the various embodiments of the invention mayalso be employed, with binding of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7 expressing cells to the solid support being determinedfollowing peripheral blood being passed through the column.

The levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expressing cells, levels of expression of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 and/or levels of cells with high expression of CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (defined as CCR2^(hi),CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi), CXCR2^(hi) or CCR7^(hi))may be determined relative to a suitable control. When diagnosing arespiratory condition, a threshold level of cells, level of expressionof CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 and/or level ofcells with high expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7 (defined as CCR2^(hi), CCR1^(hi), CCR3^(hi), CCR5^(hi),CXCR1^(hi), CXCR2^(hi) or CCR7^(hi)) may be set at or over which apositive diagnosis is made. This threshold may be determined based uponmeasuring levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expressing cells, levels of expression of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 and/or levels of cells with high expression of CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (defined as CCR2^(hi),CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi), CXCR2^(hi) or CCR7^(hi)) insamples obtained from diseased patients and comparing these levels withlevels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressingcells, levels of expression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7 and/or levels of cells with high expression of CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (defined as CCR2^(hi), CCR1^(hi),CCR3^(hi), CCR5^(hi), CXCR1^(hi), CXCR2^(hi) or CCR7^(hi)) in samplesobtained from healthy subjects.

In certain embodiments, a respiratory disorder is diagnosed on the basisof levels of chemokine receptor expressing cells. A positive diagnosismay be made in subjects based upon the presence of greater than about10%, greater than about 20%, greater than about 30%, greater than about40%, greater than about 50%, greater than about 55%, greater than about60%, greater than about 65%, greater than about 70%, greater than about75%, greater than about 80%, greater than about 85%, greater than about90%, greater than about 95%, or more chemokine receptor expressing cellsin the sample, as a percentage of total cells in the sample. In otherembodiments, a respiratory disorder is diagnosed on the basis of thepresence of a about a 1.2 fold or greater increase, such as about a 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase inchemokine receptor expressing cells, relative to healthy controls.

In specific embodiments, sarcoidosis is diagnosed on the basis of levelsof CCR1 or CCR7 expressing cells. A positive diagnosis may be made insubjects based upon the presence of greater than about 10%, greater thanabout 15% or greater than about 20% CCR7 expressing central memory Tcells in the sample, as a percentage of total cells in the sample. Apositive diagnosis may be made in subjects based upon the presence ofgreater than about 40%, greater than about 45%, greater than about 50%,greater than about 55%, greater than about 60%, greater than about 65%,greater than about 70%, greater than about 75%, greater than about 80%or greater than about 85% or more CCR7 expressing T cells in the sample,as a percentage of total cells in the sample. A positive diagnosis maybe made in subjects based upon the presence of greater than about 60%,greater than about 65%, greater than about 70%, greater than about 75%,greater than about 80%, greater than about 85% or greater than about 90%CCR1 expressing monocytes in the sample, as a percentage of total cellsin the sample. Sarcoidosis may also be diagnosed on the basis of thepresence of a about a 1.2 fold or greater increase, such as about a 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase inthe specific chemokine receptor expressing cells, relative to healthycontrols.

In certain embodiments, progression of a respiratory disorder, which maybe in the context of a treatment regime, is monitored on the basis oflevels of chemokine receptor expressing cells at different time points.Progression of a respiratory disorder may be indicated in subjects basedupon an increase of greater than about 10%, such as an increase ofgreater than about 15%, greater than about 20%, greater than about 25%,greater than about 30%, greater than about 35%, greater than about 40%,greater than about 45%, greater than about 50%, greater than about 55%,greater than about 60%, greater than about 65%, greater than about 70%,greater than about 75% or more chemokine receptor expressing cells inthe sample, as a percentage of total cells in the sample, compared to asample taken from the same subject at an earlier time point. In otherembodiments, progression of a respiratory disorder is confirmed on thebasis of the presence of a about a 1.2 fold or greater increase, such asabout a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greaterfold increase in chemokine receptor expressing cells, relative to asample taken from the same subject at an earlier time point.

In specific embodiments, sarcoidosis is monitored on the basis of levelsof CCR1 or CCR7 expressing cells such as CCR7 expressing central memoryT, CCR7 expressing T cells or CCR1 expressing monocytes. Progression ofthe disease, which may be in the context of a treatment regime, may beindicated in subjects based upon the presence of an increase of greaterthan about 10%, such as greater than about 15%, greater than about 20%,greater than about 25%, greater than about 30%, greater than about 35%,greater than about 40%, greater than about 45%, greater than about 50%,greater than about 55%, greater than about 60%, greater than about 65%,greater than about 70%, greater than about 75% or more chemokinereceptor expressing cells in the sample, as a percentage of total cellsin the sample, compared to a sample taken from the same subject at anearlier time point.

Regression or successful treatment may be monitored based upon similardecreases over various time points. For example, regression orsuccessful treatment may be indicated in subjects based upon a decreaseof about 10%, such as a decrease of about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about60%, about 65%, about 70%, about 75% or more chemokine receptorexpressing cells in the sample, as a percentage of total cells in thesample, compared to a sample taken from the same subject at an earliertime point. In other embodiments, regression of respiratory disorders isconfirmed on the basis of the presence of a about a 1.2 fold or greaterdecrease, such as about a 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or100 or greater fold decrease in chemokine receptor expressing cells,relative to a sample taken from the same subject at an earlier timepoint.

In specific embodiments, sarcoidosis is monitored on the basis of levelsof CCR1 or CCR7 expressing cells such as CCR7 expressing central memoryT, CCR7 expressing T cells or CCR1 expressing monocytes. Regression orsuccessful treatment of the disease may be made in subjects based upon adecrease of about 50%, such as such as a decrease of about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95% or more CCR1 or CCR7 expressing cells in the sample, as apercentage of total cells in the sample or by a decrease of about 10%,such as a decrease of about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75% or more chemokine receptor expressing cells in thesample, as a percentage of total cells in the sample, compared to asample taken from the same subject at an earlier time point.

Suitable software is freely available (such as the R project forstatistical computing) to perform the necessary statistical analysis ofthe data obtained to calculate a useful threshold. The threshold may beset to maximize sensitivity and/or specificity of the test. Performanceof the test in these respects may be measured by plotting a receiveroperating characteristics (ROC) curve (sensitivity versus specificity).The area under the curve provides an indication of the overallperformance of the test. Thus, once thresholds have been set fordiagnosing the condition, a separate control experiment does notnecessarily have to be run each time a sample is tested. Ratherreference can simply be made to the pre-existing thresholds to determinethe diagnosis. However, in certain embodiments, the sample is testedtogether with a control sample taken from a healthy subject to provide acomparator based upon essentially identical experimental conditions. Thetest sample is generally tested in parallel with the control sample. Thetest sample level of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expressing cells, levels of expression of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 and/or levels of cells with high expression of CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (defined as CCR2^(hi),CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi), CXCR2^(hi) or CCR7^(hi))can then be compared with that of the control sample to make thediagnosis. A control sample from a disease patient may also be tested incertain embodiments. Reference to controls permits relative levels(“high”, “low” etc.) of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expressing cells in the test sample to be readily identified and thesignificance thereof interpreted. Reference to controls also permitsrelative levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expression (“high”, “low” etc.) within the cell population to bedetermined and the significance thereof interpreted. Such determinationmay, for example, indicate the average levels of CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7 expression per cell in the test sample.

Thus, in specific embodiments, high or higher levels of one or more ofCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells orhigh or higher levels of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 expression, for example average CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 expression per cell, or high or higherlevels of one or more of CCR2^(hi), CCR1^(hi), CCR3^(hi), CCR5^(hi),CXCR1^(hi), CXCR2^(hi) and/or CCR7^(hi) cells correlate with activedisease or more active disease. Similarly, lower or low levels of one ormore of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressingcells, or low or lower levels of one or more of CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7 expression, for example average CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression per cell, or low orlower levels of one or more of CCR2^(hi), CCR1^(hi), CCR3^(hi),CCR5^(hi), CXCR1^(hi), CXCR2^(hi) and/or CCR7^(hi) cells may correlatewith a lack of active inflammation or disease. This may be defined as“less active disease”. It can readily be envisaged that control samplesmay be assessed and levels of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7 expressing cells, levels of expression of CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 and/or levels of cells with highexpression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (definedas CCR2^(hi), CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi), CXCR2^(hi) orCCR7^(hi)) determined across the range of severities of respiratoryconditions. This may assist in correlating the levels of CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, levels ofexpression of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 and/orlevels of cells with high expression of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 (defined as CCR2^(hi), CCR1^(hi), CCR3^(hi),CCR5^(hi), CXCR1^(hi), CXCR2^(hi) or CCR7^(hi)) in the test sample withthe relative severity of the condition.

When monitoring progression of, or monitoring treatment of respiratoryconditions, in particular sarcoidosis and Chronic Obstructive PulmonaryDisease (COPD), the control samples may be taken from the subject at anearlier time point. They may, however, be based upon known referencevalues as discussed above. Thus, relative levels of CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, relative levels ofCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression includingrelative levels of average CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/orCCR7 expression per cell or relative levels of CCR2^(hi), CCR1^(hi),CCR3^(hi), CCR5^(hi), CXCR1^(hi), CXCR2^(hi) and/or CCR7^(hi) cells maybe with reference to samples taken from the same subject at a differentpoint in time. In certain embodiments, decreased levels of one or moreof CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells,decreased relative levels of one or more of CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7 expression including decreased relative levelsof average CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressionper cell, or decreased relative levels of one or more of CCR2^(hi),CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi), CXCR2^(hi) and/or CCR7^(hi)cells correlate with successful treatment. The treatment may be anysuitable treatment, but in specific embodiments is a treatment accordingto the various embodiments of the invention.

When monitoring progression of respiratory conditions, in particularsarcoidosis and Chronic Obstructive Pulmonary Disease (COPD), increasedlevels of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/orCCR7 expressing cells increased relative levels of one or more of CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expression includingincreased relative levels of average CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 expression per cell or increased relative levels ofone or more of CCR2^(hi), CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi),CXCR2^(hi) and/or CCR7^(hi) cells may indicate the progression ofcondition or disease. Thus, if levels of one or more of CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells, levels ofexpression of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/orCCR7 and/or levels of cells with high expression of one or more of CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 (defined as CCR2^(hi),CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi), CXCR2^(hi) or CCR7^(hi))are increased in a sample taken later than a sample from the samepatient this may indicate progression of the condition.

Since the levels of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2and/or CCR7 expressing cells, levels of one or more of CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 expression or levels of one or more ofCCR2^(hi), CCR1^(hi), CCR3^(hi), CCR5^(hi), CXCR1^(hi), CXCR2^(hi)and/or CCR7^(hi) cells are diagnostically relevant, determining suchlevels in a sample obtained from a subject may influence treatmentselection for that subject. Accordingly, in certain embodiments theinvention provides a method of selecting a suitable treatment forrespiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD) comprising determining:

a) the levels of one or more of the chemokine receptor CCR2, CCR1, CCR3,CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells

b) levels of expression of one or more of CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7; and/or

c) levels of cells with high expression of one or more of CCR2, CCR1,CCR3, CCR5, CXCR1, CXCR2 and/or CCR7

in a sample obtained from a subject, wherein high levels of one or moreof CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressing cells,high levels of expression of one or more of CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7 or high levels of cells with high expression ofCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 or increased levels ofone or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7expressing cells compared to control, increased levels of expression ofone or more of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 comparedto a control or increased levels of cells with high expression of one ormore of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 compared to acontrol, result in selection of a treatment as defined herein fortreatment of the respiratory conditions, in particular sarcoidosis andChronic Obstructive Pulmonary Disease (COPD). In certain embodiments,the chemokine receptor expressing cells are high chemokine receptorexpressing cells, in particular, high CCR2, CCR1, CCR3, CCR5, CXCR1,CXCR2 and/or CCR7 expressing cells. In specific embodiments, the cellscomprise monocytes, in particular CCR1 and/or CCR2 expressing monocytes.In other embodiments the cells comprise lymphocytes, specifically Tlymphocytes such as central memory T cells, in particular CCR7expressing T cells.

In specific embodiments, a respiratory disorder is treated on the basisof measuring levels of chemokine receptor expressing cells. Thus, atreatment according to the various embodiments of the invention may beapplied based upon the presence of greater than about 10%, greater thanabout 20%, greater than about 30%, greater than about 40%, greater thanabout 50%, greater than about 55%, greater than about 60%, greater thanabout 65%, greater than about 70%, greater than about 75%, greater thanabout 80%, greater than about 85%, greater than about 90%, greater thanabout 95%, or more chemokine receptor expressing cells in the sample, asa percentage of total cells in the sample. In other embodiments, arespiratory disorder is treated according to the various embodiments ofthe invention on the basis of the presence of a about a 1.5 fold orgreater increase, such as about a 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50or 100 or greater fold increase in chemokine receptor expressing cells,relative to healthy controls.

In specific embodiments, sarcoidosis is treated on the basis of levelsof CCR1 or CCR7 expressing cells. Thus, a treatment according to thevarious embodiments of the invention may be applied based upon thepresence of greater than about 10%, greater than about 15% or greaterthan about 20% CCR7 expressing central memory T cells in the sample, asa percentage of total cells in the sample. Thus, a treatment accordingto the various embodiments of the invention may be applied based uponthe presence of greater than about 40%, greater than about 45%, greaterthan about 50%, greater than about 55%, greater than about 60%, greaterthan about 65%, greater than about 70%, greater than about 75%, greaterthan about 80% or greater than about 85% or more CCR7 expressing T cellsin the sample, as a percentage of total cells in the sample. Thus, atreatment according to the various embodiments of the invention may beapplied based upon the presence of greater than about 60%, greater thanabout 65%, greater than about 70%, greater than about 75%, greater thanabout 80%, greater than about 85% or greater than about 90% CCR1expressing monocytes in the sample, as a percentage of total cells inthe sample. Sarcoidosis may also be treated on the basis of the presenceof a about a 1.2 fold or greater increase, such as about a 1.5, 2, 2.5,3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in thespecific chemokine receptor expressing cells, relative to healthycontrols.

For the avoidance of doubt, all embodiments described in respect of themethods of the various embodiments of the invention apply to theseaspects mutatis mutandis and are not repeated for reasons ofconciseness. Specifically, respiratory conditions, in particularsarcoidosis and Chronic Obstructive Pulmonary Disease (COPD) may beindicated in conjunction with one or more of the following indicators:The diagnosis of sarcoidosis may be made based upon clionical findings;x-ray, biopsy, s-Ca, ACE activity. COPD diagnosis may also be made byclinical findings; X-ray spirometry and blood gas analysis. In specificembodiments, the sample is a peripheral blood sample.

The methods and medical uses of the various embodiments of the inventionthus can be tailored to the need of individual patients or groups ofpatients on the basis of the various diagnostic methods of the variousembodiments of the invention. By removing from the circulation one ormore of CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 expressingcells, such as monocytes, macrophages and lymphocytes, in particularmonocytes, upregulated in various inflammatory conditions associatedwith respiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD), an important factor in theinflammatory process of such conditions can be controlled.

The various embodiments of the invention will now be described in moredetail by reference to the following non-limiting embodiments andexamples:

DESCRIPTION OF THE FIGURES

FIGS. 1a, 1b & 1 c—the binding of biotinylized MIP-1α by CD4+, CD8+T-cells and CD14+ monocytes respectively, obtained from peripheral bloodof a healthy donor;

FIGS. 2a, 2b & 2 c—the binding of biotinylized MCP-1 by CD4+, CD8+T-cells and CD14+ monocytes respectively, obtained from peripheral bloodof a healthy donor;

FIGS. 3a, 3b & 3 c— the binding of IL-8 by by CD4+, CD8+ T-cells andCD16+ monocytes respectively, obtained from peripheral blood of ahealthy donor

FIG. 4a —binding of MCP-1 to monocytes (dashed line) in peripheral bloodtaken from IBD patients. The graph represents a summary of four tests.

FIG. 4b —binding of CCR2-antibody to monocytes (line) in peripheralblood taken from IBD patients. The graph represents a summary of fourtests.

FIG. 5a —binding of eotaxin to neutrophils/eosinophils (dashed line) inperipheral blood. The graph represents a summary of four tests.

FIG. 5b —binding of CCR3-antibody to neutrophils/eosinophils (line) inperipheral blood. The graph represents a summary of four tests.

FIG. 6—The plastic house and top showing the distribution plate (2) andsafety filter units (3 and 4).

FIG. 7—The overall leukapheresis system.

FIG. 8—The pump with air detector and optical detector (4).

FIG. 9a —Results of in vitro depletion tests performed on the bMCP-1coupled matrix showing ability to eliminate CCR2-expressing cells fromblood from three healthy donors.

FIG. 9b —Results of in vitro depletion tests performed on thebiotinylated RANTES coupled matrix showing ability to eliminatechemokine receptor-expressing cells from peripheral blood taken from ahealthy donor.

FIG. 9c —Results of in vitro depletion tests performed on thebiotinylated eotaxin coupled matrix showing ability to eliminateCCR3-expressing cells from blood from a healthy donor.

FIG. 10—Sequence and biotinylation, via a spacer group, of matureprotein MCP-1 derivative containing Gln to pyroGlu modification.

FIG. 11—Sequence and biotinylation, via a spacer group, of matureprotein MCP-1 derivative containing Gln to pyroGlu modification and Metto Norleu substitution.

FIG. 12—Sequence and biotinylation, via a spacer group, of truncatedMCP-1 derivative containing Met to Norleu substitution.

FIG. 13—Alignment of MCP-1 and MCP-5 amino acid sequences.

FIG. 14—Sequence and biotinylation, via a spacer group, of (C-terminal)truncated MCP-5 derivative containing Ile to Lys modification.

FIG. 15—Sequence and biotinylation, of RANTES derivative.

FIG. 16—Sequence and biotinylation, via a spacer group, of matureprotein eotaxin derivative containing C-terminal amide.

FIG. 17—Example of gating criteria for CCR2 expressing monocytes

FIG. 18—Frequency of CCR1 expressing monocytes in 20 healthy controlsand 2 patients with sarcoidosis. The expression of chemokine receptorsand specific cell markers were analysed with flow cytometry.

FIG. 19—Expression of CCR1 compared to binding of bRANTES to bloodmonocytes from a patient with sarcoidosis. The expression of chemokinereceptors, binding of chemokine, and specific cell markers were analysedwith flow cytometry.

FIG. 20—Depletion of CCR1 expressing monocytes with SepharoseStreptavidin-matrix conjugated with bRANTES. Blood cells from a healthycontrol were incubated with biotinylated chemokine-SepharoseStreptavidin-matrix. Unbound cells were retrieved by washing the matrix.The cells (After Depletion) were then analysed with flow cytometry andcompared with cells that had not been incubated withbiotinylated-chemokine-matrix (Before Depletion).

FIG. 21—Expression of CCR2 on monocytes from two patients withsarcoidosis. The expression of chemokine receptors, binding of chemokineand specific cell markers were analysed with flow cytometry.

FIG. 22—Binding of the chemokine bMCP-1 to monocytes. Bars representfrequency of MCP-1 binding monocytes and CCR2 expressing monocytes inblood from a patient with sarcoidosis. Blood was incubated withbiotinylated chemokine and analysed with flow cytometry.

FIG. 23—Depletion of CCR2 expressing monocytes with SepharoseStreptavidin-matrix conjugated with bMCP-1. Blood cells from a healthycontrol were incubated with biotinylated chemokine-SepharoseStreptavidin-matrix. Unbound cells were retrieved by washing the matrix.The cells (After Depletion) were then analysed with flow cytometry andcompared with cells that had not been incubated with bchemokine-matrix(Before Depletion).

FIG. 24a —Frequency of CCR7 expressing T cells. Bars represent frequencyof T cells that express CCR7 in 2 patients and 20 healthy controls. Theexpression of chemokine receptors and specific cell markers wereanalysed with flow cytometry. The T cells were characterized as CD3positive.

FIG. 24b —Frequency of central memory T cells in one patient withsarcoidosis. The central memory T cells were characterized as CD3positive, CD4 positive, CD45RA negative, CCR7 positive cells.

FIG. 25—Depletion of CCR7 expressing T cells with SepharoseStreptavidin-matrix conjugated with bMIP3b. Blood cells from a patientwith Sarciodosis were incubated with biotinylated chemokine-SepharoseStreptavidin-matrix. Unbound cells were retrieved by washing the matrix.The cells (After Depletion) were then analysed with flow cytometry andcompared with cells that had not been incubated with bchemokine-matrix(Before Depletion).

DESCRIPTION OF PREFERRED EMBODIMENTS

Plasma levels of MCP-1 and MIP-1α were monitored in 26 patients withactive pulmonary sarcoidosis over a two year period. During this period,the authors show that levels of these cytokines were closely related tothe clinical course of the disease. The authors conclude that plasmaMCP-1 and MIP-1α levels are useful indicators of clinical severity ofsarcoidosis, and that levels “may reflect subclinical evidence ofextrathoracic sarcoidosis and may play a role in initiating monocytemigration into the tissue”. Hashimoto S et al, Clin Exp Immunol, 1998

Serum MCP-1 levels were measured in 47 sarcoidosis patients and 10healthy controls. Chemokine levels were significantly higher in thepatient group, and more specifically, correlated positively withpatients in early disease stages. Furthermore, MCP-1 was shown to bespecifically expressed by macrophages associated with sarcoid lymphnodes. lyonaga K et al, Sarcoidosis Vasc Diffuse Lung Dis, 1998

The inflammatory cytokines TNF-α, IL-8, MCP-1, MMP9 and GRO-a weremeasured in 100 COPD patients and 50 matched healthy smokers. Thesevalues were subsequently correlated to the BODE index of COPD diseaseseverity. The largest difference in these biomarkers what observed inserum levels of MCP-1 which were significantly increased in the COPDgroup. The authors conclude that serum MCP-1 levels may be a clinicalcandidate for distinguishing between healthy smokers and patients withstable COPD. Liu S F et al, Respirology, 2009

TNF-alpha, IL-8, MMP-9, MCP-1, TIMP-1 and TIMP-2 were measured in 20COPD patients, 10 asymptomatic smokers and 10 non-smoker healthycontrols. The authors found highly reproducible and statisticallysignificant elevations of plasma IL-8 among the COPD patients comparedto the other groups. No other correlations were observed. Shaker S B etal, Clin Respir J, 2008.

It is shown herein that subjects suffering from respiratory conditionssuch as sarcoidosis exhibit increased frequency of chemokine receptorexpressing cells in the peripheral blood. Subjects with sarcoidosisexhibit increased frequency of CCR1 expressing cells such as CCR1expressing monocytes, compared to healthy controls. It is also shownherein that the CCR1 expressing cells can be removed using a suitablebinding reagent, in particular RANTES (in biotinylated form) immobilizedon a suitable matrix. Similarly, it is shown herein that the monocytesalso express CCR2. The CCR2 expressing monocytes can be depleted insarcoidosis patients using a suitable binding reagent, in particularMCP-1, in biotinylated form, immobilized on a suitable matrix. It isalso shown herein that subjects suffering from respiratory conditionssuch as sarcoidosis exhibit increased frequency of CCR7 expressing cellssuch as CCR7 expressing lymphocytes, and also central memory T cells,compared to healthy controls. It is also shown herein that the CCR7expressing cells can be removed using a suitable binding reagent, inparticular MIP3b (in biotinylated form) immobilized on a suitablematrix.

On this basis the inventors have selected a range of chemokine receptorsto use as targets for treatment according to the methods of theinvention.

Examples 1 to 9

Materials and Methods

Isolation of Peripheral Blood Leukocytes.

Heparinized peripheral blood from healthy blood donors or inflammatorybowel disease (IBD) patients was fixed with 4% paraformaldehyde for 4minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloridesolution and washed twice in FACS buffer to obtain a suspension of bloodleukocytes.

Chemokines.

The leukocytes were incubated for 30 min in the dark at 4° C. withbiotinylated and Alexa647 Fluor® labelled chemokine (CCL5, CCL2, CXCL8)(in concentrations 10 ng/μL and 50 ng/μL). The cells were then washedwith FACS-buffer and analyzed by flow cytometry. All chemokines used inthe Examples were provided by Almac Sciences Scotland Ltd, Edinburgh,Scotland.

Flow Cytometry Assay.

The flow cytometry assay was performed on a two laser FACS Caliburcytometer (BD Immunocytometry systems, San Jose, Calif., USA). Tenthousand cells were counted and analysed in each sample. For dataanalyses, Cell Quest Pro software from Becton Dickinson was used.

Example 1

Binding of monocytes to MIP-1α. In the experiment with biotinylatedMIP-1α it was found that about 90% of the monocytes obtained fromperipheral blood of healthy donors had bound to the cytokine after 30min of incubation (FIG. 1c ), whereas CD4+ and CD8+ lymphocytes had notbound (FIGS. 1a and 1b ).

Example 2

Binding of monocytes to MCP-1. In the experiment with biotinylated MCP-1it was found that about 90% of the monocytes obtained from peripheralblood of healthy donors had bound to the cytokine after 30 min ofincubation (FIG. 2a ), whereas CD4+ and CD8+ lymphocytes had not bound(FIGS. 2b and 2c ).

Example 3

Affinity of blood cells to biotinylated IL-8. In FIG. 3 the binding tobiotinylated IL-8 (CXCL8) of CD4+ lymphocytes (FIG. 3a ), CD8+lymphocytes (FIG. 3b ) and CD16+ neutrophils (FIG. 3c ) obtained fromhealthy donors is shown. After 30 min of incubation all CD16+neutrophils bound to IL-8. In contrast no binding was observed with CD4+lymphocytes and CD8+ lymphocytes.

Example 4

Monocytes were investigated for their expression of CCR2 (FIG. 4b ) andtheir ability to bind MCP-1 (FIG. 4a ). CCR2 expression was noted an allmonocytes with the majority of monocytes expressing high levels, usingan anti-CCR2 antibody (FIG. 4b ). The MCP-1 binding to monocytes shownin FIG. 2a corresponds to the CCR2^(hi) expressing population shown inFIG. 4b . Thus, MCP-1 binds favourably to CCR2^(hi) expressing cells.

Example 5

Neutrophils/eosinophils were investigated for their expression of CCR3,(FIG. 1b ) and their ability to bind eotaxin (FIG. 5a ). CCR3,expression was noted in all neutrophils/eosinophils with the majority ofneutrophils/eosinophils expressing high levels, using an anti-CCR3,antibody (FIG. 5b ). The eotaxin binding to neutrophils/eosinophilsshown in FIG. 5a corresponds to the CCR3^(hi) expressing populationshown in FIG. 5b . Thus, eotaxin binds favourably to CCR3^(hi)expressing cells.

Example 6

Preparation of a chemokine column for blood cell apheresis. Tostreptavidin cross-linked agarose (ProZyme, San Leandro, Calif., U.S.A.)beads in the range from 75 μm to 300μ suspended (200 ml, ˜50%, v/v) inan aqueous solution of 25 mM sodium phosphate (pH 7.0) and 150 mM NaClwas added a solution of 75 μg biotinylated MIP-1α (Almac Sciences) inthe same buffer at 22° C. and slowly stirred by hand for 3 min. Afterstanding for another 20 min, the support was filtered off, washed thricewith neutral aqueous sodium phosphate/sodium chloride and filled into aglass column (i.d. 25 mm, length 12 cm).

Example 7

Separation of monocytes from peripheral blood of a healthy donor withthe chemokine column of Example 5. Heparinized peripheral blood from ahealthy male donor was analyzed by flow cytometry for CD4+ lymphocytes,CD8+ lymphocytes and CD14 monocytes. 100 ml of the blood was filteredthrough the column at a rate of about 8 ml per min and washed with FACSbuffer. The filtered blood was analyzed for the same cells. It was foundthat about 95% of the monocytes had been retained by the column whereasmore than 90% each of CD4+ and CD8+ lymphocytes had been recovered.

Example 8

Preparation of streptavidin conjugated magnetic beads complexed withbiotinylated MIP-1α. An aqueous suspension of streptavidin conjugatedmagnetic beads (MagCellect Streptavidin Ferrofluid, 1 ml; R&D Systems,Minneapolis, Minn., U.S.A.) was mixed with 30 μg of MIP-1α (AlmacSciences) in 50 ml of 25 mM sodium phosphate (pH 7.0) and 150 mM NaCland slowly stirred for 1 hour. The particles were washed thrice with 20ml portions the same solvent and stored in suspension at 4° C.

Example 9

Separation of CD14+ monocytes from peripheral blood of a healthy donorwith the streptavidin magnetic beads of Example 7. 100 ml of heparinizedblood from the healthy donor of Example 7 was mixed with thestreptavidin conjugated magnetic beads complexed with biotinylatedMIP-1α and slowly stirred for 40 min. The particles were separated fromthe blood by a magnetic separator, and the blood analyzed for CD14+monocytes and CD4+ and CD8+ lymphocytes. While essentially no CD14+monocytes could be detected, CD4+ and CD8+ lymphocytes were present inroughly the original amounts.

Example 10—Tailored Leukapheresis

Column Design and Properties

Introduction

Apheresis is an established treatment used for depletion of bloodcomponents, such as antibodies, low-density lipoproteins (LDL) and bloodcells. Leukapheresis is the apheresis treatment used for removal ofwhite blood cells, leukocytes. The patient is connected to anextracorporeal blood circulating system; the blood is drawn from a veinin one arm, passed through a column device and returned into the otherarm of the patient. Side effects of leukapheresis treatments are varyingfrom mild events like headache, dizziness, hypotension, palpitation andflush seen in 0.1 to 5% of treated patients.

The Column

The column is intended to be used as a leukapheresis treatment forrespiratory conditions, in particular sarcoidosis and ChronicObstructive Pulmonary Disease (COPD). It will specifically remove CCR2,CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7-expressing leukocytes, inparticular monocytes, through the use of a binding reagent containingresin, exploiting the CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/orCCR7-chemokine interaction. The column consists of three combinedcomponents, the plastic house, the streptavidin (SA) Sepharose™ BigBeads matrix and one or more biotinylated chemokine bound to the matrix.The treatment is conducted using the same techniques as a standardapheresis procedure.

The Plastic House (FIG. 6)

The plastic house, designed to keep a continuous blood flow through thematrix, consists of a transparent body and red-coloured top. The top hasa distribution plate (2) at the inflow site (1) to spread the bloodevenly over the entire matrix area. The plate is the first safetybarrier preventing larger particles flowing through the column and intothe patient. Safety filter units (3 and 4) are placed at the inflow (1)and outflow (5) sites of the plastic housing. The safety filter unitcontains three filters designed to be a robust barrier and stop allparticles larger than blood cells passing through the column. Theplastic housing design is shown in FIG. 4. The design with safetyfilters (3 and 4) at both ends of the column device will minimize therisk of leakage of particles into the patient, including in the eventthat the device is placed up side down with the blood flow in theopposite direction to that anticipated.

Streptavidin Sepharose™ BigBeads

The second component in the device is the affinity matrix calledstreptavidin Sepharose™ BigBeads (Sepharose™ GE Healthcare, Sweden).Sepharose™ is a cross linked, beaded-form of agarose, which is apolysaccharide extracted from seaweed. Sepharose™ and agarose arecommonly used as column matrices in biomedical affinity techniques. Itis chosen for its optimal distribution capacity and can provide a largeavailable area for affinity binding.

Binding Reagent

Coupled to the matrix is the third component of the device, one or morebinding reagents that bind specifically to CCR2, CCR1, CCR3, CCR5,CXCR1, CXCR2 and/or CCR7. One or more chemokines may be employed. Thesepeptides may be synthetic, engineered versions of the human chemokine,which are truncated and biotinylated, but retain binding activity to theCCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 receptor. Bybiotinylating the engineered chemokine, it is able to bind to thestreptavidin molecules in the Sepharose™ matrix. The biotin-streptavidinbinding is known be one of the strongest biological interactions with aKd in the order of 4×10⁻¹⁴ M. The calculated ratio ofstreptavidin:biotin binding sites in the column is 10:1. Therefore, thecoupling between the matrix and chemokine will be immediate, minimisingthe risk of chemokine decoupling from the matrix.

The Apheresis System

To conduct the leukapheresis the following components are needed; thecolumn, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

The Circuit

The system is illustrated in FIG. 7. The patient (1) is connected to theextracorporeal circuit via sterile Venflon needles to veins in the rightand the left arms. A saline bag (3) is also connected and the salinesolution is pumped with an ACD pump (2). Blood is drawn from one arm ofthe patient through the sterile tubing system by the blood pump (4) andpassed through the column (6) and back to the patient. The tubing systemis connected to the column via standard dialysis luer-lock couplings.The couplings on the column are colour-coded for correct assembly; redtubing for inflow to the red column top and blue tubing for outflow backto the patient. An air detector (8) is present. Inlet pressure (5) andPven sensors (7) are employed to monitor the pressure in the circuit.

The 4008 ADS Pump

An apheresis pump, from Fresenius Medical Care, monitors the patient'sinflow and outflow, the pressure in the extracorporeal circulation andcan discriminate air by a bubble catcher and air detector. A clotcatcher filter is placed inside the bubble catcher. The pump also has anoptical detector to distinguish between light, e.g. saline solution orair present in the tubing system and dark e.g. blood present in thetubing system.

A schematic diagram of the pump, showing the air detector and opticalfilter is shown in FIG. 8. If the pump system detects air bubbles andoptical fluctuations or if extracorporeal pressure values are out of theset range, then the pump stops immediately and a visual/audible alarmare emitted.

Legend for FIG. 8:

-   -   1. Monitor    -   2. Holder for waste bag    -   3. Modules (left to right—Blood pump, ACD pump, Air detector)    -   4. Reserve places for further modules    -   5. Absorber holder    -   6. Drip detector    -   7. IV pole

Preparation of the Patient

The patient will be administered anticoagulants prior to each treatmentsession. A sterile saline solution with 5000 IE Heparin will be used forpriming the extracorporeal system, thereafter a bolus injection with4000 IE Heparin will be added into the circuit at the start of eachtreatment session.

Leukapheresis Time and Flow Rate

The apheresis system should be operated at a flow rate of 30-60 ml/min.A treatment is finalised after 1800 mL of blood has been circulated.

Storage Conditions

The column devices should be stored between 1 and 25° C. avoidingfreezing and more elevated temperatures. Stability data >3 monthsindicate no difference in functionality over time or by temperature(room temperature and refrigerated). The columns will be kept inrefrigerated conditions until use. Mechanical damage as those resultingfrom violent vibrations and trauma should be avoided. Column storedoutside of these recommendations should not be used.

Transport Conditions

The column devices will be transported under refrigerated condition,avoiding freezing and more elevated temperatures. Mechanical damage suchas those resulting from violent vibrations and trauma should be avoided.

In-Vitro Depletion of Target Cell Populations

To investigate the ability to eliminate CCR2-expressing cells, in vitrotests have been performed on the bMCP-1 coupled matrix. Blood wascollected from blood donors and passed through the column devicecontaining bMCP-1 coupled matrix. Blood samples were taken before andafter column passage and analyzed by flow cytometry (FACS) for thedepletion of CCR2-expressing cells.

The results demonstrate significant depletion of the target populationCCR2-expressing monocytes post matrix perfusion. Depletion tests wereperformed on blood from three healthy donors. The results are shown inFIG. 9 a.

The in-vitro results demonstrate a specific reduction of up to 80% ofthe CCR2-expressing cells by the column. Notably, individuals with fewerCCR2 expressing cells initially achieved lower depletion. The remaininglevels of monocytes were around 20-30% in each case, irrespective of thestarting point. Non-CCR2-expressing cells remained unaffected (data notshown).

To investigate the ability to eliminate CCR1, 3 and 5-expressing cells,in vitro tests have been performed on the biotinylated RANTES coupledmatrix. Blood was collected from blood donors and passed through thecolumn device containing biotinylated RANTES coupled matrix. Bloodsamples were taken before and after column passage and analyzed by flowcytometry (FACS) for the depletion of CCR1, 3 or 5-expressing cells.

The RANTES molecule was synthesized by Almac. The amino acid sequence ofthe biotinylated RANTES molecule is set forth as SEQ ID NO: 16:

H2N- SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEKS-CO2H

This molecule has the naturally occurring methionine at position 67replaced with lysine to facilitate biotinylation at position 67.

The side-chain of Lys 67 was directly biotinylated to given the proteinprimary structure shown in FIG. 15. The protein was folded anddisulphide bonds formed between the first and third cysteine in thesequence and between the 2nd and 4th cysteines.

The results demonstrate significant depletion of the target populationchemokine receptor-expressing cells post matrix perfusion. Depletiontests were performed on blood from a healthy donor. The results areshown in FIG. 9 b.

The in-vitro results demonstrate a specific reduction of around 20% ofthe chemokine receptor-expressing cells by the column. Non-CCR1, 3 and5-expressing cells remained unaffected (data not shown).

In-Vitro Depletion of Target Cell Populations

To investigate the ability to eliminate CCR3-expressing cells, in vitrotests have been performed on the eotaxin coupled matrix. Blood wascollected from blood donors and passed through the column device(including a magnetic separator) containing eotaxin coupled matrix (MACSbeads). Blood samples were taken before and after column passage andanalyzed by flow cytometry (FACS) for the depletion of CCR3-expressingcells.

The results demonstrate significant depletion of the target populationCCR3-expressing neutrophils/eosinophils post matrix perfusion. Depletiontests were performed on blood from a healthy donor. The results areshown in FIG. 9 a.

In conclusion, the in-vitro results demonstrate a specific reduction ofaround 25% of the CCR3-expressing cells by the column.Non-CCR3-expressing cells remained unaffected (data not shown).

Example 11—MCP1 Derivatives

MCP-1 has been produced with residue 75 as the site of biotinylation onthe chemokine (numbering based upon the mature protein having the aminoacid sequence of SEQ ID NO: 2). Biotinylation permits immobilization ofMCP-1 on a solid support (via a biotin-avidin interaction). The basicamino acid sequence of MCP-1, including a 23 amino acid leader sequenceis set forth as SEQ ID NO: 1,

MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCPKEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

The amino acid sequence of the mature protein is set forth as SEQ ID NO:2,

QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQKWVQDSMDHL DKQTQTPKT

The inventors have determined that chemokines may display improvedbinding properties where the chemokine is biotinylated via a spacergroup. The spacer may prevent the biotin group from impacting on thebinding affinity of the chemokine.

Thus, MCP-1 derivatised at the ε-amino side chain functionality of Lys75with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be3,6-dioxoaminooctanoic acid. The Gln at the N-terminus of the proteinsis subject to pyroGlu formation under physiological conditions. Thus thefirst glutamine (Gln1) of the sequence will be substituted withpyroglutamine. The molecule will be synthesised as a C-terminal amide(via synthesis on an amide linker). The molecule is shown schematicallyin FIG. 10.

A biotinMCP-1 Met to Nleu analogue will also be synthesised. The singlemethionine within the sequence will be altered to Norleucine, tomitigate against oxidation of this residue during the chain assembly andimprove stability of the final product. This molecule is shownschematically in FIG. 11.

Once synthesised, the activity of the various biotinMCP-1 derivativeswill be determined in cell-based assays. In particular, agonist andantagonist properties will be determined in aequorin functionalcell-based assay on human CCR2 receptor.

Example 12—Synthesis of a CCR2 Antagonist Biotin MCP-1 which Binds tothe Receptor without Activation

Antagonist Activity (J-H Gong and I. Clark-Lewis, J. Exp. Med., 1995,181, 63) has been shown for an MCP-1 derivative truncated at theN-terminus. In particular, deletion of residues 1-8, results in bindingto CCR2 with Kd 8.3 nM. This protein was unable to cause chemotaxis ofCCR2 positive cells. (inhibition of chemotaxis IC50 20 nM)

The amino acid sequence of the truncated version is set forth as SED IDNO:3:

VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHLDKQTQTPKT

A derivative of this truncated version will be synthesised comprisingresidues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleusubstitution and derivatised at the ε-amino side chain functionality ofLys75 with PEG-Biotin (TFA salt). This molecule is shown schematicallyin FIG. 12. The PEG spacer will be 3,6-dioxoaminooctanoic acid.

Once synthesised, the activity of the various biotinMCP-1 derivativeswill be determined in cell-based assays. In particular, agonist andantagonist properties will be determined in aequorin functionalcell-based assay on human CCR2 receptor.

Example 13—Demonstrate Removal of CCR2 Expressing Cells Using anAlternative Chemokine Ligand to MCP-1

CCR2 also binds chemokines MCP-2, MCP-3, MCP-4, MCP-5, and HCC-4 inaddition to MCP-1. MCP-5 only binds CCR2 and should be selective in itsremoval of CCR2 expressing cells. MCP5 is a mouse chemokine shown tochemotact human CCR2 cells with EC50<3 nM.

The full length amino acid sequence, including the signal peptide, isset forth as SEQ ID NO: 4

MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPREAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

The amino acid sequence of N-terminal processed MCP-5 chemokine is 82amino acids long and is set forth as SEQ ID NO: 5

GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEKWVKNSINHLD KTSQTFILEP SCLG

An amino acid sequence alignment suggests that MCP-5 has a C-terminalextension when compared to the amino acid sequence of MCP-1. The resultsof this alignment are shown in FIG. 13. On this basis a C-terminaltruncated version of MCP-5 will be synthesised. This truncated versionwill comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 6:

GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEKWVKNSINHLD KTSQTFIL

In the truncated version, Ile75 to be substituted with Lys, set forth asSEQ ID NO: 7:

GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEKWVKNSINHLD KTSQTFKL

Following substitution, the substituted version will be biotinylated atposition 75, a lysine or other suitable residue such as ornithine ordiaminopropanoic acid via A PEG spacer (3,6-dioxoaminooctanoic acid).The protein will be synthesised on an amide linker to yield a C-terminalamide derivative. This molecule is shown schematically in FIG. 14.

Example 14—Eotaxin Derivatives

Eotaxin has been produced with residue 73 (thought to be a lysine) asthe site of biotinylation on the chemokine (numbering based upon themature protein having the amino acid sequence of SEQ ID NO: 9).Biotinylation permits immobilization of eotaxin on a solid support (viaa biotin-avidin interaction). The basic amino acid sequence of eoxtaxin,including a 23 amino acid leader sequence (signal peptide) is set forthas SEQ ID NO: 8,

MKVSAALLWL LLIAAAFSPQ GLAGPASVPT TCCFNLANRK IPLQRLESYR RITSGKCPQKAVIFKTKLAK DICADPKKKW VQDSMKYLDQ KSPTPKP

The amino acid sequence of the mature protein is set forth as SEQ ID NO:9,

GPASVPT TCCFNLANRK IPLQRLESYR RITSGKCPQK AVIFKTKLAK DICADPKKKWVQDSMKYLDQ KSPTPKP

The inventors have determined that chemokines may display improvedbinding properties where the chemokine is biotinylated via a spacergroup. The spacer may prevent the biotin group from impacting on thebinding affinity of the chemokine.

Thus, eotaxin derivatised at the ε-amino side chain functionality ofLys73 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacerwill be 3,6-dioxoaminooctanoic acid. The molecule will be synthesised asa C-terminal amide (via synthesis on an amide linker) to avoiddiketopiperazine formation during the synthesis. The molecule is shownschematically in FIG. 16.

A biotin eotaxin Met to Nleu analogue will also be synthesised. Thesingle methionine within the sequence will be altered to Norleucine, tomitigate against oxidation of this residue during the chain assembly andimprove stability of the final product.

Once synthesised, the activity of the various eoxtaxin derivatives willbe determined in cell-based assays. In particular, agonist andantagonist properties will be determined in functional cell-based assayon human CCR3 receptor.

Once synthesised, the activity of the various biotinMCP-5 derivativeswill be determined in cell-based assays. In particular, agonist andantagonist properties will be determined in functional cell-based assayon human CCR2 receptor.

Examples 15 to 21—Chemokine Synthesis—General Protocols

Assembly:

Chemical synthesis of chemokines was performed using standard Fmoc solidphase peptides synthesis (SPPS) techniques on an ABI 433 peptidesynthesiser. DIC (0.5 M in DMF) and OxymaPure (0.5 M in DMF) were usedfor activation, acetic anhydride (0.5 M in DMF) for capping, and 20%piperidine in DMF for Fmoc deprotection. Rink Amide resin was utilisedfor the generation of C-terminal amide chemokines and Wang resin forC-terminal acid chemokines. After assembly, the resin was washed withDMF and DCM and then dried in vacuo.

Removal of Dde Protection:

The Dde protecting group was removed by treatment of resin with asolution of 2.5% hydrazine in DMF (200 ml) over a 2 hour period. Theresin was then washed with DMF.

Labelling Steps:

1. Couple Fmoc-8-amino-3,6-dioctanoic Acid (PEG)

Resin was swollen in DMF and then a solution ofFmoc-8-amino-3,6-dioctanoic acid (0.38 g, 1 mmol), DIC solution (2 ml,0.5 M in DMF) and OxymaPure solution (2 ml, 0.5 M in DMF) was added. Themixture was sonicated for 3 hours and then washed with DMF.

2. Capping

The resin was capped with acetic anhydride solution (0.5 M in DMF, 10ml) for 5 minutes and then washed with DMF.

3. Fmoc Deprotection

Fmoc deprotection was carried out by treatment with 20% piperidine inDMF solution (2×50 ml) for 15 minutes each. The resin was washed withDMF.

4. Couple Biotin-OSu

A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 μl) in DMF (10ml) was added to the resin and the mixture was sonicated for 3 hours.The resin was washed thoroughly with DMF and DCM then dried in vacuo.

Cleavage:

Dry resin was treated with TFA (10 ml) containing a scavenger cocktailconsisting of TIS (500 μl), thioanisole (500 μl), water (500 μl), DMS(500 μl), EDT (250 μl), NH₄I (500 μg) and phenol (500 μg) and themixture was stirred at room temperature for 5 hours. The solution wasfiltered into cold ether and the resin rinsed with TFA. The precipitatedpeptide was centrifuged, washed with ether, centrifuged and lyophilised.

Purification Protocol:

The crude peptide was purified by reverse phase HPLC (RP-HPLC) using aJupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimisedgradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrilecontaining 0.1% TFA].

Folding Protocol:

Pure peptide (10 mg) was dissolved into 6M GnHCl (16 ml) and thenrapidly diluted to 2M GnHCl concentration by the addition of 50 mM TRISpH 8.5 (84 ml) containing 0.3 mM GSSG and 3 mM GSH. The mixture wasstirred at room temperature for 24 hours and then analysed by RP-HPLC(Jupiter C18, 250×4.6 mm column, 10-60% B over 30 minutes. Purificationby RP-HPLC using an optimised gradient afforded the desired product.

Example 15—BiotinMCP-1 (CCL2)

Target Molecule: MCP-1 derivatised at the ε-amino side chainfunctionality of Lys(75) with PEG-Biotin (TFA salt)

Modifications: Human MCP-1 corresponding to residues 1-76, is initiallyexpressed as 99 amino acids comprising the chemokine fold, and a 23amino acid signal peptide which is cleaved off. The Gln at theN-terminus of the protein is subject to pyroGlu formation underphysiological conditions. Thus Gln1 of the sequence was substituted withpyroglutamine to prevent mixed species of N-terminal Gln and pyroGlubeing generated. This improves the yield of synthesis and ensures ahomogeneous chemokine preparation through column manufacture and use.The naturally occurring lysine at position 75 was modified throughbiotinylation on the resin. A PEG spacer was incorporated between theε-amino functionality and the biotin.

The linear amino acid sequence (SEQ ID NO: 10) is shown, prior toattachment of the PEG spacer and biotin molecules at amino acid 75 (K):

H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT-NH₂

X=pyroGlu or Gln

The engineered MCP-1 sequence was assembled on a solid support (RinkAmide resin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

SEQ ID NO: 11 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN

X1=pyroGlu or Gln

X75=K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 75 to facilitatesite-specific labelling at this position of the protein. Subsequentremoval of the ivDde protecting group, followed by coupling of the PEGspacer and Biotin, was carried out as described in the general protocolsection. Cleavage, purification and folding protocols were carried outas described to furnish the desired active chemokine.

SEQ ID NO: 12 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPXT-NH₂

X1=pyroGlu or Gln

X75 is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, optionally K(PEG-Biotin)

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinMCP-1: obtained=9032.8 Da; expected 9034.4 Da.

Functional Assay Data:

biotinMCP-1 was tested for agonist activity in an Aequorin assay againsthCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was reported. c.f. EC50for recombinant native MCP-1 is 3.1 nM.

Example 16—BiotinRANTES (CCL5)

Target Molecule: RANTES derivatised at the ε-amino side chainfunctionality of Lys(67) with Biotin (TFA salt)

Modifications: Human RANTES corresponding to residues 1-68, is initiallyexpressed as 91 amino acids comprising the chemokine fold, and a 23amino acid signal peptide which is cleaved off. The single methionine(Met67) within the sequence was mutated to lysine, to mitigate againstoxidation of this residue during the chain assembly, which was observedduring the synthesis of the natural sequence derivative. This Met to Lyssubstitution provided a lysine at position 67 which was modified throughbiotinylation on the resin.

The linear amino acid sequence (SEQ ID NO: 16) is shown, prior toattachment of the biotin molecule at amino acid 67 (K):

H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEKS-OH

The engineered RANTES sequence was assembled on a solid support (Wangresin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEXS-RESIN

X is K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 67 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 17).Subsequent removal of the ivDde protecting group, followed by couplingof the Biotin, was carried out as described in the general protocolsection. Cleavage, purification and folding protocols were carried outas described to furnish the desired active chemokine (SEQ ID NO: 18).

H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSLEXS-OH

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG (e.g. K(Biotin))

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

Functional Assay Data:

BiotinRANTES was tested for agonist activity in an Aequorin assayagainst hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 17—BiotinMCP-2 (CCL8)

Target Molecule: MCP-2 derivatised at the s-amino side chainfunctionality of Lys(75) with PEG-Biotin (TFA salt)

Modifications: Human MCP-2 corresponding to residues 1-76, is initiallyexpressed as 99 amino acids comprising the chemokine fold, and a 23amino acid signal peptide which is cleaved off. The Gln at theN-terminus of the protein is subject to pyroGlu formation underphysiological conditions. Thus Gln1 of the sequence was substituted withpyroglutamine to prevent mixed species of N-terminal Gln and pyroGlubeing generated. This improves the yield of synthesis and ensures ahomogeneous chemokine preparation through column manufacture and use.The naturally occurring lysine at position 75 was modified throughbiotinylation on the resin. A PEG spacer was incorporated between theε-amino functionality and the biotin.

The linear amino acid sequence (SEQ ID NO: 13) is shown, prior toattachment of the PEG spacer and biotin molecules at amino acid 75 (K):

H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLKP-NH₂

X=pyroGlu or Gln

The engineered MCP-2 sequence was assembled on a solid support (RinkAmide resin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLXP-NH₂

X1=pyroGlu or Gln

X75=K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 75 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 14).Subsequent removal of the ivDde protecting group, followed by couplingof the PEG spacer and Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine(SEQ ID NO: 15):

H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVRDSMKHLDQIFQNLXP-NH₂

X1=pyroGlu or Gln

X75=an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

Functional Assay Data:

biotinMCP-2 was tested for activity in an Aequorin assay against hCCR2b,(Euroscreen) and was shown to be a partial agonist with an EC50 value of50.9 nM. c.f. EC50 for recombinant native MCP-2 is 23.5 nM (partialagonist).

Example 18—BiotinMIP-313 (CCL19)

Target Molecule: MIP-3β derivatised at the s-amino side chainfunctionality of Lys(78) with Biotin (TFA salt)

Modifications: Human MIP-3β corresponding to residues 1-77, is initiallyexpressed as 98 amino acids comprising the chemokine fold, and a 21amino acid signal peptide which is cleaved off. An additional lysine wasinserted at the C-terminus, at position 78, and modified throughbiotinylation on the resin.

The linear amino acid sequence (SEQ ID NO: 19) is shown, prior toattachment of the biotin molecule at amino acid 78 (K):

H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH₂

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated (e.g.K-biotin), optionally via a spacer molecule such as PEG, in particularK(PEG-Biotin)

The engineered MIP-3β sequence was assembled on a solid support (RinkAmide resin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-RESIN

X is FmocLys(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 78 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 20).Subsequent removal of the ivDde protecting group, followed by couplingof the Biotin, was carried out as described in the general protocolsection. Cleavage, purification and folding protocols were carried outas described to furnish the desired active chemokine (SEQ ID NO: 21).

H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH₂

X is K(Biotin)

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinMIP-3β: obtained=9148.8 Da; expected 9149.7 Da.

Functional Assay Data:

biotinMip-3β was tested for agonist activity in an Aequorin assayagainst hCCR7, (Euroscreen) and an EC50 value of 11.0 nM was reported.c.f. EC50 for recombinant native MIP-3β is 1.6 nM.

Example 19—BiotinIL-8 (CXCL8)

Target Molecule: IL-8 derivatised at the s-amino side chainfunctionality of Lys(78) with PEG-Biotin (TFA salt)

Modifications: Human IL-8 corresponding to residues 1-77, is initiallyexpressed as 99 amino acids comprising the chemokine fold, and a 22amino acid signal peptide which is cleaved off. An additional lysine wasinserted at the C-terminus at position 78, and modified throughbiotinylation on the resin. A PEG spacer was incorporated between theα-amino functionality and the biotin.

The linear amino acid sequence (SEQ ID NO: 22) is shown, prior toattachment of the PEG spacer and biotin molecules:

H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSX-NH₂

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin)

The engineered IL-8 sequence was assembled on a solid support (RinkAmide resin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSX-RESIN

X is K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 78 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 23).Subsequent removal of the ivDde protecting group, followed by couplingof the PEG spacer and Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine(SEQ ID NO: 24):

H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSX-NH₂

X is K(PEG-Biotin)

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinIL-8: obtained=9416.9 Da; expected 9417.0 Da.

Functional Assay Data:

BiotinIL-8 was tested for agonist activity in an Aequorin assay againsthCXCR1, (Euroscreen) and an EC50 value of 18.9 nM was reported. c.f.EC50 for recombinant native IL-8 is 4.2 nM.

Example 20—BiotinIL-8 (6-78)

Target Molecule: IL-8 (6-78) derivatised at the s-amino side chainfunctionality of Lys(78) with PEG-Biotin (TFA salt)

Modifications: Truncated form of IL-8 corresponding to residues 6-77,the first five N-terminal residues have been removed and an additionallysine was inserted at the C-terminus at position 78, and modifiedthrough biotinylation on the resin. A PEG spacer was incorporatedbetween the s-amino functionality and the biotin.

The linear amino acid sequence (SEQ ID NO: 25) is shown, prior toattachment of the PEG spacer and biotin molecules:

H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSX-NH₂

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG

The engineered IL-8 sequence was assembled on a solid support (RinkAmide resin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSX-RESIN

X is K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 78 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 26).Subsequent removal of the ivDde protecting group, followed by couplingof the PEG spacer and Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine(SEQ ID NO: 27):

H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENSX-NH₂

X is K(PEG-Biotin)

Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) dataof purified folded biotinIL-8 (6-78): obtained=8880.50 Da; expected8880.4 Da.

Functional Assay Data:

BiotinIL-8 (6-78) was tested for agonist activity in an Aequorin assayagainst hCXCR1, (Euroscreen) and an EC50 value of 6.1 nM was reported.c.f. EC50 for recombinant native IL-8 is 4.2 nM.

Example 21—BiotinEotaxin (CCL11)

Target Molecule: Eotaxin derivatised at the s-amino side chainfunctionality of Lys(73) with PEG-Biotin (TFA salt)

Modifications: Human eotaxin corresponding to residues 1-74, isinitially expressed as 97 amino acids comprising the chemokine fold, anda 23 amino acid signal peptide which is cleaved off. The naturallyoccurring lysine at position 73 was modified through biotinylation onthe resin. A PEG spacer was incorporated between the ε-aminofunctionality and the biotin.

The linear amino acid sequence (SEQ ID NO: 28) is shown, prior toattachment of the PEG spacer and biotin molecules at amino acid 73 (K):

H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP-NH₂

X is an amino acid residue that can be biotinylated, such as lysine,ornithine or diaminopropionic acid and optionally is biotinylated,optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin).

The engineered eotaxin sequence was assembled on a solid support (RinkAmide resin), using Fmoc protocols for solid-phase peptide synthesis asdescribed in the general protocols section:

H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP-NH₂

X is K(ivDde)

FmocLys(ivDde)-OH was incorporated as residue 73 to facilitatesite-specific labelling at this position of the protein (SEQ ID NO: 29).Subsequent removal of the ivDde protecting group, followed by couplingof the PEG spacer and Biotin, was carried out as described in thegeneral protocol section. Cleavage, purification and folding protocolswere carried out as described to furnish the desired active chemokine(SEQ ID NO: 30):

H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPXP-NH₂

X is K(PEG-Biotin)

Electrospray ionisation with tandem mass spectrometry (ESi-TOF-MS) dataof purified folded biotinEotaxin: obtained=8731.3 Da; expected 8731.3Da.

Functional Assay Data:

biotinEotaxin was tested for activity in an Aequorin assay againsthCCR3, (Euroscreen) and was shown to be an antagonist with an EC50 valueof 211.8 nM. c.f. EC50 for recombinant native eotaxin is 10.7 nM(agonist).

Example 22—Diagnosis and Treatment of Sarcoidosis

Materials and Methods

1. Flow Cytometric Analysis of Peripheral Blood

Peripheral blood from patients and healthy controls was collected inheparin tubes. The red blood cells were lysed using Fix Buffer(Phosphate Buffer Saline (PBS) citrate with 4% paraformaldehyde) forfour minutes at 37° C. and Lysing buffer (PBS with 10 mM Tris and 160 mMNH4Cl, pH 7.5) for 15 min at 37° C. The cells were washed in PBS with 2%Bovine Growth Serum, incubated with 10% human serum for 15 min at roomtemperature (RT) and stained with antibodies (Table 2) at 4° C. for 30min. The cells were analysed by flow cytometry on a FACS Canto flowcytometer with the FACSDiva software (BD Biosciences).

TABLE 2 List of antibodies for flow cytometric analysis. AntibodyFluorophore Supplier CCR1 Alexa flour 647 Biolegend CCR2 PerCPCy5.5Biolegend CCR7 PerCpCy5.5 Biolegend CD4 V500 BD CD3 Horizon V450 BDStreptavdin APC BD CD14 FITC Beckman Coulter CD45RA PECy7 BD

2. Chemokine Binding Test

Peripheral blood from patients and healthy controls was collected inheparin tubes. The red blood cells were lysed using Fix Buffer(Phosphate Buffer Saline (PBS) citrate with 4% paraformaldehyde) forfour minutes at 37° C. and Lysing buffer (PBS with 10 mM Tris and 160 mMNH4Cl, pH 7.5) for 15 min at 37° C. The cells were washed in PBS with 2%Bovine Growth Serum, incubated with 10% human serum 15 min at roomtemperature (RT) and stained with cell specific antibodies together withbiotinylated chemokine (1 μM) or the corresponding chemokine receptorantibody at 4° C. for 30 min (Table 1). The biotinylated chemokine wasdetected via the interaction between biotin and a fluorophore conjugatedStreptavidin. The samples were analysed by flow cytometry on a FACSCanto flow cytometer with the FACSDiva software (BD Biosciences).

3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

Cells were prepared from peripheral blood (section 1). 1 mL SepharoseBigBeads matrix conjugated with 0.4 mg/mL Streptavidin (GE Healthcare)was washed in 50 mL PBS and added to a 5 mL polystyrene tube (BDFalcon™). Biotinylated chemokine was added to the tube and incubated for20 min at RT to enable immobilization of the chemokine on the matrix viathe biotin-streptavidin interaction. Next, the cells were added to thechemokine-matrix and incubated for 20 min at RT. The cells that did notbind to the matrix were removed by washing the matrix with PBS in asterile 40 um nylon filter (BD Falcon™ Cell Strainer). The flow throughcells were stained with antibodies (Table 2), analysed by flow cytometryand compared with cells from peripheral blood that had not beenincubated with the chemokine-matrix.

Results and Discussion

White blood cells from 2 patients with sarcoidosis were analysed for theexpression of chemokine receptors with flow cytometry. The patientsexhibited increased frequency of monocytes that expressed the receptorCCR1 based upon flow cytometry data and binding of an anti-CCR1 antibody(FIG. 18).

The ligand for CCR1 is the chemokine RANTES that also binds to CCR5expressed on T cells. RANTES is expressed in the lungs where it mediatesmigration of inflammatory cells. The monocytes bind biotinylated RANTESto the same extent as the chemokine receptor expression (FIG. 19).

The CCR1 expressing monocytes could be efficiently depleted withbRANTES-conjugated Sepharose Streptavidin Matrix (FIG. 20).

In addition to CCR1, the monocytes express the chemokine receptor CCR2(FIG. 21), based upon flow cytometry data and binding of an anti-CCR2antibody.

The ligand for CCR2 is MCP-1 that mediate migration of monocytes ininflammation. In accordance with the CCR2 expression, biotinylated MCP-1(bMCP-1) could bind to blood monocytes from a sarcoidosis patient (FIG.22).

The CCR2 expressing monocytes could be depleted with bMCP1-conjugatedSepharose Streptavidin Matrix (FIG. 23).

The sarcoidosis patients exhibit an increased frequency of circulating Tcells that express the chemokine receptor CCR7 (FIG. 24a ), based uponflow cytometry data and binding by an anti-CCR7 antibody. Furthermore,the frequency of central memory T cells, which are characterized as CCR7positive, is increased in sarcoidosis. (FIG. 24b ). Central memory Tcells contribute to inflammation by mounting a fast and strong immuneresponse the second time the inflammation is triggered, and may beresponsible for relapsing sarcoidosis.

The ligand for CCR7 is MIP3b. The CCR7 expressing T cells could beefficiently depleted with bMIP3b-conjugated Sepharose StreptavidinMatrix (FIG. 25)

We conclude that the frequency of CCR1 expressing monocytes and T cellsthat express CCR7 is enhanced in Sarcoidosis. The CCR2 receptor isexpressed on monocytes from sarcoidosis patients to the same extent asin the healthy controls, but the CCR2 expressing cells could differ intheir pro-inflammatory profile in the patients compared to healthycontrols. Both monocytes and T cells bind the chemokines thatcorresponded with the expression of the chemokine receptor, and could beefficiently depleted with the corresponding biotinylatedchemokine-Sepharose Streptavidin-matrix.

The present various embodiments of the invention is not to be limited inscope by the specific embodiments described herein. Indeed, variousmodifications of the various embodiments of the invention in addition tothose described herein will become apparent to those skilled in the artfrom the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims. Moreover, all embodiments described herein are considered to bebroadly applicable and combinable with any and all other consistentembodiments, as appropriate.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

The invention claimed is:
 1. A method for treating a respiratory condition in a subject in need thereof, the method comprising applying peripheral blood from the subject to an apheresis column loaded with a solid support comprising one or more binding reagents capable of specifically binding to chemokine receptor CCR2 immobilized directly or indirectly on the support, whereby one or more cells expressing the chemokine receptor CCR2 are removed from the peripheral blood of the subject, wherein the applied blood is returned to the subject, whereby the respiratory condition is treated, and wherein the respiratory condition is sarcoidosis or Chronic Obstructive Pulmonary Disease (COPD).
 2. The method of claim 1 wherein the binding reagent is an agonist or an antagonist of CCR2.
 3. The method of claim 1, wherein the binding reagent is an antibody or a chemokine.
 4. The method of claim 3, wherein the chemokine is selected from MCP-1, MCP-2, MCP-3, MCP-4 and MCP-5 and the chemokine receptor is CCR2.
 5. The method of claim 4, wherein the chemokine is MCP-1 or MCP-5.
 6. The method of claim 1, wherein the one or more cells are monocytes, eosinophils, or T lymphocytes.
 7. The method of claim 1, wherein the subject has increased levels of expression of CCR2 as compared to a subject that does not have a respiratory condition.
 8. The method of claim 1, wherein 20-50% of the subject's blood is applied to the column in a single treatment. 